PhilipAllenThesis

!

!

Modulating!Trim21!to!Ameliorate!Disease!

Symptoms!in!Patients!with!Autoimmune!Diseases!!

!

Thesis&by&

!

!

Philip!Allen!Dy!!

!

In!Partial!Fulfilment!of!the!Requirements!

For!the!Degree!of!

Bachelor!of!Science!in!Bio–Molecular!Science!

14th&–&May&–&2015&

!

!

Presented&to&the&

School!of!Biological!Sciences!

College!of!Sciences!and!Health!

Dublin!Institute!of!Technology!!

!

!

!

Supervised!By:!Dr.!Joan!Ní!Gabhann!

Honorary!Lecturer!in!Biochemistry!

Molecular!and!Cellular!Therapeutics!

Royal!College!of!Surgeons!in!Ireland!

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Acknowledgments!

!I!would!like!to!give!special!thanks!to!my!supervisors!on!this!project,!Dr.!Joan!Ní!

Gabhann!and!Dr.!Jay!Chandanshive.!Throughout!the!project,!both!of!you!have!

been!wonderful!mentors!and!have!given!me!unlimited!support,!for!which!I!am!

immensely!grateful.!

!

I!would!also!like!to!extend!my!thanks!to!Professor!Caroline!Jefferies,!Professor!

Conor!Murphy,!Dr.!Qistina!Pilson,!Dr.!Siobhán!Smith,!Shane!O’Grady,!Leah!Arkins!

and!all!the!researchers!in!the!MCT!Molecular!Immunology!Department!for!all!

their!kind!help!and!support!everyone!have!aided!me!during!my!time!in!your!lab.!

Thank!you!for!making!me!feel!welcome!and!I!wish!each!and!every!one!in!MCT!

great!success!in!their!respective!future!endeavours.!Also,!Dr.!Claire!Wynne’s!

efforts!with!helping!me!get!around!RCSI!and!for!her!kind!donation!of!293Ts,!

thank!you!!

!

Lastly,!to!my!family!and!friends!who!have!been!there!since!the!beginning!and!all!

the!encouragements!and!neverXending!support!for!which!I!am!exceptionally!

thankful!!

!

!

!

!!

ii!!

List!of!Abbreviations!Abbreviations, Description,AECG! AmericanXEuropean!Consensus!Group!ALS! Amyotrophic!Lateral!Sclerosis!AML! Acute!Myeloid!Leukaemia!BCA! Bicinchoninic!Acid!BS! Binding!Site!CC! CoiledXCoil!CD! Cyclodextrin!CD80! Cluster!of!Differentiation!80!cDNA! Complementary!DNA!CFC! Californian!Fox!Criteria!CFTR! Cystic!Fibrosis!Transmembrane!Conductance!Regulator!CRC! Colorectal!Cancer!CSF2! Colony!Stimulating!Factor!2!CXCL3! Chemokine!(CXXXC!motif)!Ligand!3!DB! Database!DC! Drug!Compound!DCM! Dilated!Cardiomyopathy!DGCR8! DiGeorge!Syndrome!Critical!Region!Gene!8!DMD! Duchenne!Muscular!Dystrophy!DMEM! Dulbecco’s!Modified!Eagle!Medium!DMSO! Dimethyl!Sulphoxide!DNA! Deoxyribonucleic!Acid!DSF! Differential!Scanning!Fluorimetry!E.coli& Escherichia&coli&E2F3! E2F!Transcription!Factor!3!ECC! European!Community!Criteria!EDTA! Ethylenediaminetetraacetic!Acid!EV! Empty!Vector!FBS! Foetal!Bovine!Serum!GHD! Growth!Hormone!Deficiency!HCC! Hepatocellular!Carcinoma!HEK293XT! Human!Embryonic!Kidney!293XT!cells!HGT! Hepatocyte!Growth!Factor!HIV! Human!Immunodeficiency!Virus!HLA! Human!Leukocyte!Antigen!IFN! Interferon!IGF1! InsulinXlike!Growth!Factor!1!

!

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IL! Interleukin!IL10! Interleukin!10!IL17D! Interleukin!17D!IRF! Interferon!Regulatory!Factor!ITP! Idiopathic!Thrombocytopenic!Purpura!JAK3! Janus!Kinase!3!LB! Lysogeny!Broth!MACC1! Metastasis!Associated!in!Colon!Cancer!1!MALT! MucosaXAssociated!Lymphoid!Tissue!MHC! Major!Histocompatibility!Complex!miRNA/miR! microRNA!MS! Multiple!Sclerosis!MyD88! Myeloid!Differentiation!Primary!Response!88!NaOH! Sodium!Hydroxide!NCBI! National!Centre!for!Biotechnology!Information!NPSLE! Neuropsychiatric!Lupus!NSAIDS! Nonsteroidal!AntiXInflammatory!Drugs!OSCC! Oral!Squamous!Cell!Carcinoma!PBMC! Peripheral!Blood!Mononuclear!Cells!PBS! Phosphate!Buffered!Saline!PCR! Polymerase!Chain!Reaction!PEG! Polyethylene!Glycol!PRR! Pathogen!Recognition!Receptor!pSS! Primary!Sjögren’s!Syndrome!qPCR! Qualitative!Polymerase!Chain!Reaction!RA! Rheumatoid!Arthritis!RISC! RNAXInduced!Silencing!Complex!RNA! Ribonucleic!Acid!RPMI!1640! Roswell!Park!Memorial!Institute!1640!RT! Room!Temperature!SCD! Sickle!Cell!Disease!SEM! Standard!Error!of!the!Mean!SLE! Systemic!Lupus!Erythematosus!SOCS1! Suppressor!of!Cytokine!Signalling!1!SS! Sjögren’s!Syndrome!TAE! TrisXAcetateXEDTA!TBS! TrisXBuffered!Saline!TE! TrisXEDTA!TFA! Trifluoroacetic!Acid!TLR! TollXlike!Receptors!

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Tm! Annealing!Temperature!TNF! Tumour!Necrosis!Factor!TRIM! Tripartite!MotifXContaining!Protein!TRIM21! Tripartite!MotifXContaining!Protein!21!TS! Target!Score!UV! Ultraviolet!VC! Vehicle!Control!

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List!of!Tables!!

TABLE!1.1:!SJÖGREN'S!SYNDROME!–!REVISED!AECG!CRITERIA.!ADAPTED!FROM!(VITALI!ET!AL.,!2002).!..............!3!

TABLE!1.2:!AMERICAN!COLLEGE!OF!RHEUMATOLOGY!CRITERIA!FOR!THE!DIAGNOSIS!OF!SYSTEMIC!LUPUS!

ERYTHEMATOSUS.!ADAPTED!FROM!(TSOKOS,!2011).!............................................................................................!4!

TABLE!1.3:!NEUROPSYCHIATRIC!SYNDROMES!OBSERVED!IN!SYSTEMIC!LUPUS!ERYTHEMATOSUS.!ADAPTED!FROM!

(MANSON!AND!RAHMAN,!2006).!...............................................................................................................................!7!

TABLE!1.4:!ENVIRONMENTAL!FACTORS!THAT!MAY!BE!RELEVANT!IN!THE!PATHOGENESIS!OF!SYSTEMIC!LUPUS!

ERYTHEMATOSUS.!ADAPTED!FROM!(MOK!AND!LAU,!2003).!...............................................................................!12!

TABLE!2.1:!PROCEDURE!FOR!MAKING!UP!THE!BCA!STANDARDS!AS!PER!MANUFACTURER’S!GUIDELINES.!..............!28!

TABLE!3.1:!CONCENTRATIONS!OF!PLASMID!DNA!DETERMINED!BY!NANODROP.!.......................................................!36!

TABLE!3.2:!%!INHIBITION!OF!LUCIFERASE!ASSAYS.!.........................................................................................................!47!

TABLE!3.3:!BCA!RESULTS!OF!THE!STANDARDS.!................................................................................................................!48!

TABLE!3.4:!PROTEIN!CONCENTRATION!OF!PROTEIN!SAMPLES!DETERMINED!BY!BCA!ASSAY.!...................................!48!

TABLE!3.5:!MIRS!OF!INTEREST!WITH!TRIM21!AS!THE!PREDICTED!GENE!TARGET!ALONG!WITH!ITS!ASSOCIATED!

DISEASES.!.......................................................................................................................................................................!55!

TABLE!3.6:!EXPRESSION!RATIO!OF!THE!STATISTICALLY!SIGNIFICANT!MIRS!OF!INTEREST!FROM!NANOSTRING!

SCREENING!BETWEEN!PATIENTS!AND!CONTROLS.!..................................................................................................!56!

TABLE!3.7:!UPREGULATED!AND!DOWNREGULATED!MIRS!OF!INTEREST.!......................................................................!56!

TABLE!3.8:!LIST!OF!PRIMER!SEQUENCES!FOR!MIRS!OF!INTEREST!AND!ITS!RESPECTIVE!GENE!TARGETS.!..................!57!

TABLE!3.9:!MRNA!CONCENTRATION!IN!PSS!PATIENTS!AND!HEALTHY!CONTROLS!USING!NANODROP!

SPECTROPHOTOMETER.!..............................................................................................................................................!60!

TABLE!6.1:!TOP!30!MIRS!THAT!ARE!OVEREXPRESSED!IN!PSS!PATIENTS!VS!SLE!FROM!NANOSTRING!SCREENING.

!........................................................................................................................................................................................!88!

TABLE!6.2:!TOP!30!MIRS!THAT!ARE!REDUCED!IN!PSS!PATIENTS!VERSUS!SLE!FROM!NANOSTRING!SCREENING.!..!89!

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List!of!Figures!!FIGURE!1.1:!EFFECTS!OF!SJÖGREN’S!SYNDROME!IN!THE!BODY.!TAKEN!FROM!(KALLEN,!2014).!................................!6!FIGURE!1.2:!COMMON!SYMPTOMS!OF!SYSTEMIC!LUPUS!ERYTHEMATOSUS,!WHICH!CAN!VARY!BETWEEN!

INDIVIDUALS.!TAKEN!FROM!(THE!LUPUS!EFFECT,!X).!..............................................................................................!8!FIGURE!1.3:!OVERVIEW!OF!THE!PATHOGENESIS!OF!SLE.!ADAPTED!FROM!(TSOKOS,!2011).!.....................................!9!FIGURE!1.4:!BIOGENESIS!OF!MIRNA.!TAKEN!FROM!(SIGMAXALDRICH,!X).!...................................................................!13!FIGURE!1.5:!THE!STRUCTURAL!DOMAINS!OF!RO52.!TAKEN!FROM!(OKE!AND!WAHRENXHERLENIUS,!2012).!......!15!FIGURE!2.1:!THE!BLUE!PUREYIELD™!CLEARING!COLUMN!IN!A!WHITE!PUREYIELDTM!MAXI!BINDING!COLUMN!ON!A!

VACXMAN®!LABORATORY!VACUUM!MANIFOLD!PORT.!..........................................................................................!21!FIGURE!2.2:!STEPS!IN!MAXIPREP!SYSTEM!(SIGMAXALDRICH,!N.D.).!..............................................................................!21!FIGURE!2.3:!ASSEMBLY!OF!MICROCENTRIFUGE!TUBE!ONTO!THE!BASE!OF!THE!ELUATOR™!VACUUM!ELUTION!

DEVICE.!..........................................................................................................................................................................!22!FIGURE!2.4:!LAYOUT!OF!A!96XWELL!PLATE!TRANSFECTION!FOR!THE!ADDITION!OF!PLASMID!DNAS.!......................!25!FIGURE!2.5:!DUAL!REPORTER!LUCIFERASE!ASSAY!SYSTEM.!.............................................................................................!26!FIGURE!2.6:!SCHEMATIC!DIAGRAM!OF!THE!REACTION!FOR!THE!BICINCHONINIC!ACIDXCONTAINING!PROTEIN!ASSAY!

(WHITE,!2007).!..........................................................................................................................................................!27!FIGURE!2.7:!SCHEMATIC!DIAGRAM!FOR!HIGH!THROUGHPUT!DYNAMIC!SCANNING!FLUORIMETRY!THERMAL!

STABILITY!ASSAY.!A,!ILLUSTRATES!THE!SAMPLE!PREPARATION!AND!B,!REPRESENTS!THE!NATIVE,!

DENATURED,!AND!AGGREGATED!STATES!OF!PROTEIN!ALONGSIDE!ITS!TYPICAL!EXOGENOUS!FLUORESCENCE!

RESPONSE!(VOLLRATH!ET!AL.,!2014).!.....................................................................................................................!29!FIGURE!2.8:!SCHEMATIC!DIAGRAM!OF!THE!STEPS!INVOLVED!IN!GEL!ELECTROPHORESIS!(PREMEDHQ,!2001).!....!32!FIGURE!2.9:!REALXTIME!PCR!MECHANISM!(SIGMAXALDRICH,!2002).!.........................................................................!35!FIGURE!3.1:!TRIM21!NEGATIVELY!REGULATES!TRIF!DRIVEN!IFNXΒ!PROMOTER!ACTIVITY.!HEK293TXCELLS!

WERE!SEEDED!AT!1X105!CELLS/ML,!200ΜL/WELL,!IN!A!96XWELL!PLATE.!CELLS!WERE!TRANSFECTED!

WITH!PLASMIDS!CONTAINING!100NG!OF!TRIF!TO!DRIVE!THE!SYSTEM,!P125,!RENILLA,!TRIM21,!AND!AN!

EV!CONTROL.!18HR!POSTXTRANSFECTION!CELLS!WERE!HARVESTED!AND!COMPOUNDS!ADDED!AND!

LUCIFERASE!ASSAY!WAS!MEASURED!FOLLOWING!6HR!INCUBATION.!A,!CYCLODEXTRIN!(CD)!VEHICLE!

CONTROL;!B,!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL;!AND!C,!SODIUM!HYDROXIDE!(NAOH)!

VEHICLE!CONTROL.!THE!ABOVE!DATA!IS!GRAPHED!FROM!THE!AVERAGE!OF!THREE!SEPARATE!EXPERIMENTS,!

±!STANDARD!ERROR!OF!THE!MEAN!SEM!(N=3).!....................................................................................................!38!FIGURE!3.2:!TRIM21!NEGATIVELY!REGULATES!TRIF!DRIVEN!IFNXΒ!PROMOTER!ACTIVITY.!HEK293TXCELLS!




LUCIFERASE!ASSAY!WAS!MEASURED!FOLLOWING!6HR!INCUBATION.!A,!3A&COMPOUND!IN!CYCLODEXTRIN!

(CD)!VEHICLE!CONTROL!WITH!DILUENTS!INCLUDING!VC!ITSELF,!PBS,!AND!STERILEXFILTERED!DMEM;!

AND!B,!3A&COMPOUND!IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL!WITH!DILUENTS!INCLUDING!VC!

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ITSELF,!PBS,!AND!STERILEXFILTERED!DMEM.!BOTH!COMPOUNDS!ARE!AT!0.05ΜM!CONCENTRATION.!THE!

ABOVE!DATA!IS!GRAPHED!FROM!THE!AVERAGE!OF!THREE!SEPARATE!EXPERIMENTS,!±!SEM!(N=3).!............!39!FIGURE!3.3:!TRIM21!NEGATIVELY!REGULATES!TRIF!DRIVEN!IFNXΒ!PROMOTER!ACTIVITY.!HEK293TXCELLS!





(CD)!VEHICLE!CONTROL!WITH!DILUENTS!INCLUDING!VC!ITSELF,!PBS,!AND!STERILEXFILTERED!DMEM;!

AND!B,!4A&COMPOUND&IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL!WITH!DILUENTS!INCLUDING!VC!

ITSELF,!PBS,!AND!STERILEXFILTERED!DMEM.!BOTH!COMPOUNDS!ARE!AT!0.05ΜM!CONCENTRATION.!THE!

ABOVE!DATA!IS!GRAPHED!FROM!THE!AVERAGE!OF!THREE!SEPARATE!EXPERIMENTS,!±!SEM!(N=3).!............!40!FIGURE!3.4:!TRIM21!NEGATIVELY!REGULATES!TRIF!DRIVEN!IFNXΒ!PROMOTER!ACTIVITY.!HEK293TXCELLS!





(CD)!VEHICLE!CONTROL;!B,!3A&COMPOUND&IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL;!AND!C,!

3A&COMPOUND!IN!TRIFLUOROACETIC!ACID!(TFA)!VEHICLE!CONTROL.!THE!ABOVE!DATA!IS!GRAPHED!FROM!

THE!AVERAGE!OF!THREE!SEPARATE!EXPERIMENTS,!±!SEM!(N=3).!*P<0.05!WAS!CONSIDERED!SIGNIFICANT.

!........................................................................................................................................................................................!42!FIGURE!3.5:!TRIM21!NEGATIVELY!REGULATES!TRIF!DRIVEN!IFNXΒ!PROMOTER!ACTIVITY.!HEK293TXCELLS!





(CD)!VEHICLE!CONTROL;!B,!4A!COMPOUND!IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL;!AND!C,!

4A&COMPOUND&IN!TRIFLUOROACETIC!ACID!(TFA)!VEHICLE!CONTROL.!THE!ABOVE!DATA!IS!GRAPHED!FROM!







(CD)!VEHICLE!CONTROL;!B,!3A&COMPOUND&IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL;!AND!C,!





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(CD)!VEHICLE!CONTROL;!B,&4A&COMPOUND&IN!POLYETHYLENE!GLYCOL!(PEG)!VEHICLE!CONTROL;!AND!C,&



!........................................................................................................................................................................................!46!FIGURE!3.8:!STANDARD!CURVE!OF!THE!BCA!STANDARDS.!.............................................................................................!48!FIGURE!3.9:!DETERMINING!PROTEIN!CONCENTRATIONS!OF!TRIM21!FOR!FURTHER!ANALYSIS!IN!THERMAL!SHIFT!

ASSAY.!............................................................................................................................................................................!51!FIGURE!3.10:!DETERMINING!PROTEIN!CONCENTRATIONS!OF!IRF3!FOR!FURTHER!ANALYSIS!IN!THERMAL!SHIFT!

ASSAY.!............................................................................................................................................................................!51!FIGURE!3.11:!DETERMINING!PERCENTAGE!OF!DMSO!USED!IN!TRIM21!PROTEIN!IN!THERMAL!SHIFT!ASSAY.!.......!52!FIGURE!3.12:!DETERMINING!PERCENTAGE!OF!DMSO!USED!IN!IRF3!PROTEIN!IN!THERMAL!SHIFT!ASSAY.!............!52!FIGURE!3.13:!ANALYSIS!OF!SERIES!A!COMPOUNDS!ON!TRIM21!PROTEIN!USING!THERMAL!DENATURATION!ASSAY.

!........................................................................................................................................................................................!53!FIGURE!3.14:!ANALYSIS!OF!SERIES!A!COMPOUNDS!ON!IRF3!PROTEIN!USING!THERMAL!DENATURATION!ASSAY.!..!53!FIGURE!3.15:!OPTIMISATION!OF!MIRX30E!AND!MIRX125A!AT!50˚,!55˚,!AND!62˚C!ANNELING!TEMPERATURES!

USING!STERILE!NUCLEASEXFREE!WATER!AND!CONTROL!CDNA!AND!1KB!DNA!LADDER!VISUALISED!BY!

SYPRORED!STAINING!ON!A!1%!AGAROSE!GEL.!........................................................................................................!58!FIGURE!3.16:!OPTIMISATION!OF!MIRX194!AND!MIRX98!AT!50˚,!55˚,!AND!62˚C!ANNEALING!TEMPERATURES!

USING!STERILE!NUCLEASEXFREE!WATER!AND!CONTROL!CDNA!AND!1KB!DNA!LADDER!VISUALISED!BY!

SYPRORED!STAINING!ON!A!1%!AGAROSE!GEL.!........................................................................................................!59!FIGURE!3.17:!OPTIMISATION!OF!THE!PREDICTED!GENE!TARGETS!OF!MIRS!OF!INTEREST!INCLUDING!CXCL3!AT!

62˚,!65˚,!AND!68˚C!ANNEALING!TEMPERATURE!AND!JAK3,!IL10,!AND!SOCS1!AT!62˚C!ANNEALING!

TEMPERATURE!USING!STERILE!NUCLEASEXFREE!WATER!AND!CONTROL!CDNA!AND!1KB!DNA!LADDER!

VISUALISED!BY!SYPRORED!STAINING!ON!A!1%!AGAROSE!GEL.!.............................................................................!59!FIGURE!3.18:!EXPRESSION!LEVELS!OF!MIRX30E!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!MIRX

30E!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.5714.!B,!MIRX30E!EXPRESSION!MINUS!THE!

OUTLIERS!WITH!A!PDVALUE&OF!0.6571.!ENDOGENOUS!RNU6B!WAS!USED!AS!REFERENCE!CONTROL!FOR!MIR!

EXPRESSION.!..................................................................................................................................................................!62!FIGURE!3.19:!EXPRESSION!LEVELS!OF!MIRX125A!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!!A,!

MIRX125A!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE&OF!0.3754.!B,!MIRX125A!EXPRESSION!MINUS!

THE!OUTLIERS!WITH!A!PDVALUE!OF!0.6667.!ENDOGENOUS!RNU6B!WAS!USED!AS!REFERENCE!CONTROL!FOR!

MIR!EXPRESSION.!.........................................................................................................................................................!62!FIGURE!3.20:!EXPRESSION!LEVELS!OF!MIRX194!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!MIRX

194!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.7023.!B,!MIRX125A!EXPRESSION!MINUS!THE!

OUTLIERS!WITH!A!PDVALUE!OF!>0.9999.!ENDOGENOUS!RNU6B!WAS!USED!AS!REFERENCE!CONTROL!FOR!

MIR!EXPRESSION.!.........................................................................................................................................................!63!

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FIGURE!3.21:!EXPRESSION!LEVELS!OF!MIRX98!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!MIRX98!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.8983.!B,!MIRX98!EXPRESSION!MINUS!THE!OUTLIERS!WITH!A!PDVALUE!OF!0.5238.!ENDOGENOU!RNU6B!WAS!USED!AS!REFERENCE!CONTROL!FOR!MIR!EXPRESSION.!..................................................................................................................................................................!63!

FIGURE!3.22:!EXPRESSION!LEVELS!OF!SOCS1!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!SOCS1!GENE!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.9319.!B,!SOCS1!GENE!EXPRESSION!MINUS!THE!OUTLIERS!WITH!A!PDVALUE!OF!0.6277.!ENDOGENOUS!18S!WAS!USED!AS!REFERENCE!CONTROL!FOR!GENE!EXPRESSION.!...............................................................................................................................................!65!

FIGURE!3.23:!EXPRESSION!LEVELS!OF!CXCL3!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!CXCL3!GENE!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.5604.!B,!CXCL3!GENE!EXPRESSION!MINUS!THE!OUTLIERS!WITH!A!PDVALUE!OF!0.7706.!ENDOGENOUS!18S!WAS!USED!AS!REFERENCE!CONTROL!FOR!GENE!EXPRESSION.!...............................................................................................................................................!65!

FIGURE!3.24:!EXPRESSION!LEVELS!OF!JAK3!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!JAK3!GENE!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.4000.!B,!JAK3!GENE!EXPRESSION!MINUS!THE!OUTLIERS!WITH!A!PDVALUE!OF!0.3333.!ENDOGENOUS!18S!WAS!USED!AS!REFERENCE!CONTROL!FOR!GENE!EXPRESSION.!..................................................................................................................................................................!66!

FIGURE!3.25:!EXPRESSION!LEVELS!OF!IL10!IN!PBMCS!OF!PSS!PATIENT!VERSUS!HEALTHY!CONTROLS.!A,!IL10!GENE!RAW!SPECTRUM!EXPRESSION!WITH!A!PDVALUE!OF!0.4000.!ENDOGENOUS!18S!WAS!USED!AS!REFERENCE!CONTROL!FOR!GENE!EXPRESSION.!........................................................................................................!66!

FIGURE!6.1:!GENE!6737!(TRIM21)!WASS!PREDICTED!TO!BE!TARGETED!BY!21!MIRNAS!IN!MIRDB.!....................!87!FIGURE!6.2:!PREDICTED!MIRNA!SITES!OF!TRIM21!ON!MRNA!3'!UTR!REGION!PRODUCED!BY!MIRWALK!AND!

OTHER!PROGRAMS.!.......................................................................................................................................................!87!

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Table!of!Contents!!

ABSTRACT!.........................................................................................................................................!1!1.0!INTRODUCTION!........................................................................................................................!2!1.1!SJÖGREN’S!SYNDROME!..............................................................................................................................!2!1.2!SYSTEMIC!LUPUS!ERYTHEMATOSUS!.......................................................................................................!2!1.3!EPIDEMIOLOGY!...........................................................................................................................................!3!1.4!SYMPTOMS!...................................................................................................................................................!5!1.5!PATHOGENESIS!...........................................................................................................................................!8!1.6!GENETIC!PREDISPOSITION!.....................................................................................................................!10!1.7!ENVIRONMENTAL!FACTORS!...................................................................................................................!11!1.8!DISTINCTIONS!BETWEEN!SS!AND!SLE!.................................................................................................!12!1.9!MICRORNAS!IN!AUTOIMMUNE!DISEASES!...........................................................................................!13!1.10!TRIM21!...................................................................................................................................................!15!1.11!AIMS!OF!THIS!STUDY!.............................................................................................................................!17!

2.0!MATERIALS!AND!METHODS!...............................................................................................!18!2.1!MATERIALS!................................................................................................................................................!18!2.1.1&General&Chemicals&and&Conditions&..........................................................................................&18!2.1.2&List&of&Suppliers&...............................................................................................................................&19!

2.2!PLASMID!PURIFICATION!.........................................................................................................................!19!2.2.1&Transformation&of&Competent&Cells&with&Plasmid&DNA&.................................................&19!2.2.2&Maxiprep&from&Escherichia&coli&................................................................................................&20!2.2.3&DNA&Purification&.............................................................................................................................&21!2.2.4&Elution&of&DNA&by&Vacuum&.........................................................................................................&22!2.2.5&DNA&quantitation&using&the&NanoDrop&1000&Spectrophotometer&...........................&22!

2.3!CELL!CULTURE!..........................................................................................................................................!23!2.3.1&Growth&and&Maintenance&of&HEK293DT&cells&......................................................................&23!2.3.2&Preparation&of&Instruments,&Culture&Medium,&and&Plates&............................................&23!2.3.3&Cell&Counting&.....................................................................................................................................&24!2.3.4&SubDculture&of&HEK293DT&cells&..................................................................................................&24!2.3.5&Transient&Transfection&.................................................................................................................&24!2.3.6&Addition&of&Compounds&................................................................................................................&25!

2.4!LUCIFERASE!ASSAY!..................................................................................................................................!26!2.5!BICINCHONINIC!ACID!(BCA)!ASSAY!.....................................................................................................!27!

xi!!

2.6!THERMAL!MELT!.......................................................................................................................................!28!2.7!BIOINFORMATICS!ANALYSIS!OF!MIR!SCREEN!DATA!..........................................................................!30!2.8!PRIMER!DESIGN!AND!OPTIMISATION!...................................................................................................!31!2.9!GEL!ELECTROPHORESIS!(1%!AGAROSE)!............................................................................................!32!2.10!ISOLATION!OF!PERIPHERAL!BLOOD!MONONUCLEATED!CELLS!(PBMCS)!.................................!33!2.11!ISOLATION!AND!QUANTITATION!OF!MRNA!.....................................................................................!34!2.12!SYNTHESIS!OF!CDNA!............................................................................................................................!34!2.13!QUANTITATIVE!PCR!(QPCR)!.............................................................................................................!35!

3.0!RESULTS!....................................................................................................................................!36!3.1!PLASMID!DNA!PURIFICATION!...............................................................................................................!36!3.2!LUCIFERASE!ASSAY!..................................................................................................................................!38!3.3!BCA!ASSAY!................................................................................................................................................!47!3.4!MELT!CURVES!...........................................................................................................................................!49!3.5!BIOINFORMATICS!.....................................................................................................................................!54!3.6!PRIMER!OPTIMISATION!...........................................................................................................................!57!3.6.1&Primer&Sequences&............................................................................................................................&57!3.6.2&Gel&Analysis&of&miRs&on&interest&+&predicted&genes&..........................................................&58!

3.7!MRNA!CONCENTRATION!IN!PSS!PATIENTS!AND!HEALTHY!CONTROLS!.........................................!60!3.8!EXPRESSION!OF!MIRS!OF!INTEREST!IN!PSS!PATIENTS!VS!HEALTHY!CONTROLS!..........................!61!3.9!EXPRESSION!OF!PREDICTED!GENE!TARGETS!OF!MIRS!OF!INTEREST!.............................................!64!

4.0!DISCUSSION!..............................................................................................................................!67!4.1!LUCIFERASE!ASSAYS!................................................................................................................................!67!4.2!THERMAL!SHIFT!ASSAY!..........................................................................................................................!70!4.3!BIOINFORMATICS!.....................................................................................................................................!71!

5.0!BIBLIOGRAPHY!.......................................................................................................................!75!

6.0!APPENDICES!............................................................................................................................!87!APPENDIX!A!......................................................................................................................................................!87!APPENDIX!B!......................................................................................................................................................!88!

!

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Abstract(Autoimmune"diseases,"in"particular"Sjögren’s"Syndrome"and"Systemic"Lupus"Erythematosus,"are"conditions"that"result"from"dysregulated"immune"responses"culminating"in"loss"of"tolerance"to"self–"antigens"and"immune>mediated"injury."In"addition"to"the"primary"pathologies"associated"with"these"diseases,"patients"can"develop"additional"complications."These"complications"can"range"in"severity"from"fatigue,"rash,"and"joint"pain"to"the"development"of"cardiovascular,"neurological,"and"renal"complications"in"addition"to"pulmonary"fibrosis,"interstitial"lung"disease"and"in"certain"cases"for"Sjögren"patients,"non>Hodgkin’s"lymphoma."To"date,"overproduction"of"inflammatory"cytokines"(IL>6,"IL>12,"IL>23"and"TNFα)"and"type"I"interferons"(IFN>α"and"IFN>β)"have"been"suggested"to"play"a"key"role"in"the"development"and"pathogenesis"of"these"autoimmune"conditions."Trim21"has"been"identified"as"key"negative"regulator"of"inflammation,"whose"expression"and"function"are"altered"in"patients"with"SLE."Trim21"is"a"negative"regulator"of"innate"immune"signalling"through"the"interaction,"ubiquitination,"and"subsequent"proteosomal"degradation"of"the"transcription"factors"IRF3"and"IRF7."By"ubiquitinating"and"targeting"IRF3"and"IRF7"for"degradation,"Trim21"effectively"turns"off"the"production"of"type"I"IFNs,"thus"preventing"overproduction"of"this"potentially"pathogenic"cytokine."Drug"compounds,"specifically"3A"and"4A,"have"been"applied"to"demonstrate"its"ability"to"increase"Trim21"activity"hence,"increased"inhibition"of"IFN>β"promoter"activity."Preliminary"results"with"4A"compounds"looked"to"be"significant"(p<0.05)."Recently,"non>coding"microRNAs"have"been"shown"to"regulate"immune"function"in"a"variety"of"autoimmune"conditions,"which"suggests"that"micro>RNA"based"strategies"hold"therapeutic"potential"in"treatment"of"autoimmune"disorders."This"project"hypothesises"that"dysregulation"of"miRNAs"in"PBMCs"of"pSS"patients"versus"healthy"controls"results"in"uncontrolled"expression"of"their"gene"targets"leading"to"disease"pathogenesis."Through"bioinformatics"analysis,"the"miRNAs"of"interest"showed"a"lot"promise"signifying"a"potential"future"role"of"microRNAs"as"novel"therapeutics.

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1.0!Introduction!Autoimmune"disorders"such"as"Sjögren’s"Syndrome"(SS)"and"Systemic"Lupus"Erythematosus"(SLE),"result"from"impairment"of"the"immune"response"leading"to"recognition"and"the"attack"of"the"patient’s"own"tissues,"and"thus"a"loss"of"selfXtolerance"(Snow"et"al.,"2003).""

1.1!Sjögren’s!Syndrome!

Henrik"Sjögren,"an"ophthalmologist"in"the"1930s,"observed"and"reported"detailed"clinical"and"histological"findings"in"women"with"chronic"xerostomia"and"keratoconjunctivitis"sicca"(Delaleu"et"al.,"2005)."It"was"previously"known"as"sicca"syndrome,"and"associated"with"low"secretions"from"both"the"lacrimal"and"salivary"gland."Sjögren’s"syndrome,"as"it"is"now"referred"to,"is"a"chronic"autoimmune"disease"that"affects"the"exocrine"glands"and"together"with"excessive"inflammation"(Bayetto"and"Logan,"2010)"are"the"main"pathophysiological"associations"essentially"leading"to"dry"eyes"and"dry"mouth."SS"primarily"affects"90%"of"women,"notably"menopausal"women,"with"a"9:1"ratio"to"men."SS"can"exist"either"as"a"primary"disorder"(pSS)"or"in"patients"associated"with"other"rheumatic"disorders,"such"as"SLE,"rheumatoid"arthritis"(RA)"or"scleroderma,"and"is"then"termed"‘secondary’"SS,"typically"as"a"late"complication.""

1.2!Systemic!Lupus!Erythematosus!

Although"the"term"“Lupus"Erythematosus”"was"introduced"by"19thXcentury"physicians"to"define"skin"lesions,"it"took"almost"100"years"to"realise"that"the"disease"is"systemic"and"spares"no"organ"and"that"it"is"caused"by"aberrant"autoimmune"response"(Duarte"et"al.,"2011)."SLE"is"a"complex"autoimmune"condition"with"heterogeneous"clinical"manifestations"of"unknown"origin"known"to"affect"virtually"every"organ"in"the"body"(YildirimXToruner"and"Diamond,"2011)."SLE"is"primarily"caused"by"autoantibodies"and"immune"complex"deposition."Similar"to"SS,"most"patients"afflicted"are"women,"especially"of"childbearing"age"with"a"9:1"ratio"commonly"reported."

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1.3!Epidemiology!

Together"with"RA,"SS"is"one"of"the"most"frequent"autoimmune"diseases"and"it"occurs"all"over"the"world."The"disease"can"appear"at"any"age,"with"a"significant"peak"incidence"between"40"and"50"years."Depending"on"the"applied"classification"criteria"the"prevalence"for"SS"reaches"from"0.5%"of"all"adult"females"when"employing"the"Californian"Fox"Criteria"(CFC)"(Fox"et"al.,"1986),"up"to"1X3%"of"total"population,"when"using"the"European"Community"Criteria"(ECC)"(Vitali"et"al.,"1993)"or"the"AmericanXEuropean"Consensus"Group"(AECG)"criteria"(Vitali"et"al.,"2002)."Recently,"the"revised"AECG"criteria"are"now"considered"the"gold"standard"for"the"classification"of"SS"(refer"to"Table,1.1)"and"have"repudiated"preceding"criteria"sets."However,"most"studies"start"from"the"assumption"of"a"prevalence"of"0.6%"of"the"total"population"(Delaleu"et"al.,"2005)."

Table!1.1:!Sjögren's!Syndrome!–!Revised!AECG!Criteria.!Adapted!from!(Vitali!et!al.,!2002).!

"Until"the"AECG"criteria"were"devised,"it"was"difficult"to"compare"different"epidemiological"studies"that"used"different"definitions"for"SS."Kivity"et&al."has"reported"prevalence"ranging"from"0.1%"to"4.8%"but"significantly"depended"on"different"geographical"and"ethnical"regions"(Kivity"et"al.,"2014)."For"example,"China"has"reported"a"prevalence"of"only"0.77%,"while"Greece"appears"to"have"an"incidence"six"times"greater"of"approximately"4.8%"(Mavragani"and"Moutsopoulos,"2010)."It"is"possible"that"the"variability"observed"might"reflect"differences"in"the"diagnostic"criteria"between"different"regions"and"ethnicities,"as"well"as"the"diverse"geographic"study"region,"which"implies"contribution"from"

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both"genetic"and"environmental"pressures,"not"to"mention"sample"size"and"gender"distribution.""The"incidence"and"prevalence"of"SLE"seems"to"be"increasing,"probably"due"to"the"identification"of"milder"cases"and"improved"survival."According"to"D’Cruz"et&al."there"is"an"obvious"difference"in"geographical"variability"in"disease"incidence"and"prevalence"(D'Cruz"et"al.,"2007)."They"reported"that"in"the"US"population,"the"entire"race"incidence"was"5.1"per"100,000"per"year"and"the"prevalence"was"52.2"per"100,000,"with"comparative"figures"of"3.8"and"26.2"in"the"UK,"and"2.9"and"28.4"in"Japan,"respectively."These"figures"are"substantially"higher"in"some"ethnic"populations,"markedly"in"individuals"of"AfricanXAmerican"or"AfricanXCaribbean"descent"(Johnson"et"al.,"1995)."However,"reliable"data"about"the"prevalence"of"SLE"are"difficult"to"come"by"due"to"variable"methods"for"data"collection"as"well"as"inconsistency"regarding"case"definition"are"known"to"be"contributing"factors"(refer"to"Table,1.2)."

Table!1.2:!American!College!of!Rheumatology!Criteria!for!the!Diagnosis!of!Systemic!Lupus!

Erythematosus.!Adapted!from!(Tsokos,!2011).!

"

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1.4!Symptoms!

Dry"eyes"and"dry"mouth"are"the"commonest"complaints"of"patients"suffering"from"SS."The"onset"is"usually"insidious"with"the"general"discomfort"typically"accompanied"by"systemic"pain"and"intense"fatigue"leading"to"an"impaired"quality"of"life"(Cornec"et"al.,"2014).""Patients"with"mild"dry"eyes"can"usually"get"away"with"selfXmedication"with"overXtheXcounter"lubricating"eye"drops."Although"many"patients"with"SS"are"still"able"to"produce"tears"with"stimulation,"it"is"inhibition"of"basal"tear"production"that"makes"the"eyes"feel"dry"(Bowman"and"Rao,"2014)."Occasionally,"dry"eyes"are"treated"with"local"application"of"cyclosporine"(ThanouXStavraki"and"James,"2008)"that"can"ameliorate"the"ocular"sicca"aspects"of"the"disease."""Deficiencies"in"the"quantity"and"quality"of"saliva"have"a"negative"impact"on"dental"and"oral"health."Dental"caries,"mucositis,"oral"candidiasis,"and"swelling"of"the"salivary"glands"are"the"most"frequent"oral"signs"of"SS"(Delaleu"et"al.,"2005)."The"most"common"cause"of"dry"mouth,"as"believed"by"Bowman"and"Rao,"are"medications"such"as"antiXdepressants,"antihistamines,"diuretics"or"betaXblockers"(Bowman"and"Rao,"2014)."Clinical"features"include"a"lack"of"obvious"saliva"in"the"mouth"and"absence"of"a"normal"‘pool’"of"saliva"underneath"the"tongue."The"simplest"solution"in"terms"of"treatment"is"to"keep"the"mouth"moistened"using"water"followed"by"a"saline"mixture.""SS"comprises"a"broad"spectrum"of"symptoms"ranging"from"organ"specific"autoimmune"exocrinopathy"to"systemic"disease"manifestations"(refer"to"Figure,1.1)."Systemic"features"of"SS"occur"in"about"twoXthirds"of"all"patients"(GarciaXCarrasco"et"al.,"2002)"and"include"joint"involvement,"Raynaud’s"phenomenon,"vasculitis,"involvement"of"the"central"and"peripheral"nervous"systems,"and"renal"and"pulmonary"involvement"(Hatron"et"al.,"2011)."In"addition,"patients"with"SS"have"a"16X"to"44Xfold"increased"risk"of"lymphoma"compared"to"the"general"population"(Theander"et"al.,"2006)"and"particularly"in"pSS"patients,"mucosaXassociated"lymphoid"tissue"(MALT)"type"B"cell"lymphoma"(Bowman"and"Rao,"

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2014)."The"prevalence"of"malignant"lymphoma"in"pSS"was"shown"to"be"stronger"than"in"SLE"or"RA"(Voulgarelis"and"Moutsopoulos,"2003).""Hatron"et&al."reported"that"pulmonary"involvement"is"also"common"in"pSS"patients,"but"only"about"10%"of"them"are"symptomatic"(Hatron"et"al.,"2011)."The"main"pulmonary"manifestations"are"sicca"cough,"and"mild"to"moderate"airways"obstruction."Pulmonary"manifestations"in"SS"typically"develop"late"in"the"course"of"disease"(Cain"et"al.,"1998)"and"are"rarely"the"presenting"feature."Pulmonary"involvement"is"associated"with"a"4Xfold"increased"mortality"risk"after"10"years"of"disease"(Stojan"et"al.,"2013)."

"

Figure!1.1:!Effects!of!Sjögren’s!syndrome!in!the!body.!Taken!from!(Kallen,!2014).!

The"clinical"features"of"SLE"are"diverse"(refer"to"Figure,1.2)."As"with"SS,"the"symptoms"of"SLE"vary"from"person"to"person."The"skin,"musculoskeletal,"and"haematologic"organs"are"commonly"involved"with"some"patients"presenting"with"

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predominantly"haematologic,"renal,"or"neuropsychiatric"manifestations"(Gill"et"al.,"2003).""Constitutional"symptoms"mainly"include"fatigue,"weight"loss,"and"fever"(in"the"absence"of"infection)"are"commonly"not"lifeXthreatening"but"have"an"impact"on"the"patient’s"quality"of"life."Renal"disease"affects"about"30%"of"patients"with"SLE"(Manson"and"Rahman,"2006),"and"remains"the"most"dangerous,"lifeXthreatening"complication."Regular"urinalysis"and"blood"pressure"monitoring"is"crucial"since"renal"involvement"is"often"asymptomatic,"especially"in"the"initial"stages.""Neuropsychiatric"lupus"(NPSLE)"is"seen"in"about"20%"of"cases"but"is"often"difficult"to"make"a"diagnosis."The"American"College"of"Rheumatology"(Rheumatology,"1999)"described"19"different"clinical"manifestations"(refer"to"Table,1.3)"but"no"single,"simple"diagnostic"test"can"be"made."

Table!1.3:!Neuropsychiatric!syndromes!observed!in!systemic!lupus!erythematosus.!Adapted!from!

(Manson!and!Rahman,!2006).!

"Skin"involvement"in"SLE"is"very"common."In"addition"to"the"classic"malar"and"discoid"rashes"seen,"more"generalised"photosensitivity"is"often"present,"and"furthermore"exposure"to"sunlight"is"known"to"trigger"systemic"disease"flares."Recurrent"crops"of"mouth"ulcers"have"also"been"found"to"be"a"feature"of"active"disease"(Manson"and"Rahman,"2006)."There"are"also"other"oral"manifestations"known"to"contribute"including"dryness"as"a"result"of"secondary"Sjögren’s"syndrome,"with"patients"also"experiencing"dryness"of"the"eyes"and"vagina."

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The"clinical"manifestations"of"SLE"are"fundamentally"the"same"in"children"and"adults."Studies"conducted"by"(Singh"et"al.,"1997;"Marini"and"Costallat,"1999)"of"children"with"SLE,"reported"that"the"most"frequent"manifestations"of"the"disease"were"fever,"rash,"arthritis,"alopecia,"and"renal"involvement."In"comparison,"children"have"a"higher"incidence"of"malar"rash,"anaemia,"leukocytopeania"(Rood"et"al.,"1999),"and"severe"manifestations"such"as"neurologic"or"renal"involvement"(Carreño"et"al.,"1999).""

"

Figure! 1.2:! Common! symptoms! of! systemic! lupus! erythematosus,! which! can! vary! between!

individuals.!Taken!from!(The!Lupus!Effect,!`).!

!

1.5!Pathogenesis!

The"pathogenesis"of"SS"has"not"been"delineated,"but"it"is"believed"that"both"immunologically"mediated"and"nonXimmune"mechanisms"contribute"significantly"(Cotrim"and"Alevizos,"2014)."Genetic"susceptibility"as"well"as"epigenetic"contributions"are"within"the"basis"of"the"disease"along"with"

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immensely"influencing"environmental"triggers."Kivity"et&al."suggested"that"genes"involved"in"innate"and"adaptive"immunity"are"crucial"for"susceptibility"(Kivity"et"al.,"2014).""SLE"is"a"multisystem"disease"(refer"to"Figure,1.3)"with"evidence"of"genetic"susceptibility,"environmental"effects,"and"disturbances"in"both"innate"and"adaptive"immunity"manifest"by"disturbances"in"apoptotic"cell"clearance,"cytokines,"BXcell"immunity,"and"TXcell"signalling"(Lisnevskaia"et"al.,"2014)."Genetic,"environmental,"hormonal,"epigenetic,"and"immunoregulatory"factors"act"either"sequentially"or"simultaneously"on"the"immune"system."The"action"of"pathogenic"factors"results"in"the"generation"of"autoantibodies,"immune"complexes,"autoreactive"or"inflammatory"T"cells,"and"inflammatory"cytokines"that"may"initiate"and"amplify"inflammation"and"damage"to"various"organs."

!

Figure!1.3:!Overview!of!the!Pathogenesis!of!SLE.!Adapted!from!(Tsokos,!2011).!

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1.6!Genetic!Predisposition!

Both"Sjögren’s"syndrome"and"systemic"lupus"erythematosus"are"considered"complex"disorders."Susceptibility"to"both"diseases"can"be"attributed"to"interplay"between"genetic"factors"and"the"environment."In"complex"diseases,"one"specific"gene"is"neither"necessary"nor"sufficient"for"disease"expression.""Genetic"factors"associated"with"SS"are"particular"human"leukocyte"antigen"(HLA)XDR"allele"subtypes"of"the"major"histocompatibility"complex"(MHC)"class"II"genes"and"specific"gene"polymorphisms"such"as"STAT4,"ILX12A,"TNIP1,"IRF5,"BLK,"and"CXCR5"(Lessard"et"al.,"2013)."Patients"of"different"ethnic"origins"exhibit"different"HLA"gene"associations."There"have"been"reports"to"suggest"that"several"families"involving"two"or"more"cases"of"Sjögren’s"syndrome"(Reveille"et"al.,"1984;"Lichtenfeld"et"al.,"1976;"Sabio"et"al.,"2001)."However,"the"level"of"genetic"contribution"is"not"known"(Bolstad"and"Jonsson,"2002),"since"there"has"not"been"genetic"linkage"studies"of"SS"families."SS"in"twins"(Scofield"et"al.,"1997;"Bolstad"et"al.,"2000)"have"also"been"described"but"the"concordance"rate"cannot"be"estimated"due"to"a"lack"of"large"cohort"studies"on"twins"with"SS."They"reported"that"twins"exhibited"a"very"similar"phenotype"with"almost"identical"clinical"manifestation,"including"dry"eyes"and"dry"mouth.""HLA"is"associated"with"the"presence"of"certain"degrees"of"diversification"of"Ro"(SSA)"and"La"(SSB)"autoantibodies"in"SS."However,"the"contribution"of"these"autoantibodies"in"SS"has"not"been"fully"understood."The"tolerance"breakdown"as"well"as"the"autoantibody"response"generation"is"still"unknown."Ultraviolet"radiation,"viral"infections"and"apoptosis"have"been"suggested"to"lead"to"undesirable"cell"surface"exposure"of"autoantigens"to"the"immune"system"(Ohlsson"et"al.,"2002)."Ro/SSA"and"La/SSB"have"been"demonstrated"in"surface"blebs"of"apoptotic"ultravioletXirradiated"keratinocytes,"which"implies"a"possible"role"in"systemic"lupus"erythematosus"(CasciolaXRosen"et"al.,"1994).""The"concordance"rate"of"SLE"in"identical"twins"is"approximately"25X50%"and"that"in"dizygotic"twins"is"around"5%"(Pisetsky,"1997)."This"suggests"that"genetic"factors"play"an"important"role"in"the"predisposition"of"the"disease."However,"

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most"cases"of"SLE"are"sporadic"without"identifiable"genetic"predisposing"factors,"implying"that"multiple"environmental"or"yet"unknown"factors"may"also"be"responsible.""Many"different"genes"contribute"to"disease"susceptibility"and"(Byrne"et"al.,"2012)"indicated"that"susceptibility"to"SLE"is"heritable."In"a"small"proportion"of"patients"(<5%),"a"single"gene"may"be"responsible."For"instance,"(Walport"et"al.,"1998)"reported"that"patients"with"homozygous"deficiencies"of"the"early"component"of"complement"are"at"risk"of"developing"SLE"or"a"lupusXlike"disease."Those"encoding"complement"components"C2"and"C4"confer"risk"for"SLE"in"certain"ethnic"groups,"but"patients"with"homozygous"C4A"null"alleles,"irrespective"of"ethnic"origin,"are"at"high"risk"of"developing"SLE."For"most"of"the"remaining"patients,"multiple"genes"are"required."It"is"estimated"that"at"least"four"susceptibility"genes"are"needed"for"the"development"of"the"disease"(Mok"and"Lau,"2003).""HLA"class"II"genes"have"also"been"extensively"studied"for"their"contribution"to"human"SLE."An"association"of"HLA"DR2"and"DR"with"SLE"is"a"common"finding"in"patients"of"different"ethnicities,"with"a"relative"risk"for"the"development"of"disease"of"approximately"two"to"five"(Pisetsky,"1997)."SLE"shows"a"strong"familial"aggregation,"with"a"much"higher"frequency"among"firstXdegree"relatives"of"patients.""

1.7!Environmental!Factors!

Exogenous"agents"such"as"EpsteinXBarr"virus"have"been"suggested"to"cause"SS"in"genetically"predisposed"individuals"(Pflugfelder"et"al.,"1993)"but"no"definite"trigger"has"yet"been"identified"in"SS."Delaleu"et&al."concluded"that"there"has"not"been"definite"exogenous"triggers"with"precise"association"with"SS"(Delaleu"et"al.,"2004)."The"long"time"span"between"disease"initiation"and"diagnosis"makes"it"particularly"difficult"to"solve"this"problem.""Although"genetic"factors"may"create"a"predisposition"towards"SLE,"the"initiation"of"the"disease"probably"results"from"several"environmental"factors"and"

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exogenous"factors"(refer"to"Table,1.4)."Certain"environmental"factors,"such"as"chemicals"and"drugs,"ultraviolet"light"(especially"UVB),"dietary"factors,"viruses,"and"circulating"oestrogen"are"probably"needed"to"precipitate"the"onset"of"SLE"(Mok"and"Lau,"2003)."

Table!1.4:!Environmental!factors!that!may!be!relevant!in!the!pathogenesis!of!systemic!lupus!

erythematosus.!Adapted!from!(Mok!and!Lau,!2003).!

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1.8!Distinctions!between!SS!and!SLE!

Since"Sjögren’s"and"Lupus"are"both"autoimmune"diseases,"there"is"a"coXexistent"association"between"the"two"conditions."Both"SS"and"SLE"are"collagen"vascular"diseases"that"can"be"accompanied"by"Ro"antibodies."Findings"report"that"a"gender"bias"preference"is"seen"where"both"primarily"affect"women"more"than"men."They"also"share"the"same"autoantibody"profile"with"similar"HLA"haplotypes"of"class"II"markers,"which"often"results"in"the"generation"of"antiXRo/La"antibodies"rather"than"from"the"diseases"themselves."Clinical"evidence"suggests"that"they"are"wholly"distinct"diseases."The"antinuclear"antibody"laboratory"diagnosis"is"more"often"positive"in"SLE"patients"vs."SS"patients."A"major"difference"between"the"two"diseases"is"the"infrequent"encounter"of"photosensitivity"often"associated"with"high"levels"of"Ro/SSA"antibodies"in"SS"patients"(Scheinfeld,"2006)."SS"does"not"respond"to"hydroxychloroquine"in"a"reproducible"fashion"whereas"SLE"usually"responds"to"hydroxychloroquine"in"a"consistent"manner"as"means"of"treatment."

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Since"the"discovery"of"microRNAs"(miRNAs),"several"papers"have"revealed"that"miRNAs"can"play"a"role"in"autoimmune"diseases."It"is"well"known"that"miRNAs"are"involved"in"the"development"of"mature"immune"cells"as"well"as"controlling"their"functions"(Carissimi"et"al.,"2009),"which"suggests"that"these"molecules"may"also"be"implicated"in"the"development"of"inflammatory"and"autoimmune"diseases.""

1.9!MicroRNAs!in!Autoimmune!Diseases!

miRs"are"small"nonXcoding"RNA"molecules"of"21X24"nucleotides"long"(Dai"et"al.,"2007)."The"genes"that"encode"miRs"comprise"1X5%"of"all"genes,"making"miRs"the"most"abundant"class"of"fineXtuning"regulators"of"diverse"biological"processes,"including"the"ability"to"control"the"expression"of"approximately"30%"of"proteinXcoding"genes"(Kapsogeorgou"et"al.,"2011)"as"well"as"regulating"immune"development"and"normal"function"and"autoimmunity"(Alevizos"and"Illei,"2010).""

"

Figure!1.4:!Biogenesis!of!miRNA.!Taken!from!(Sigma`Aldrich,!`).!

miRNA"originate"in"the"nucleus"of"the"cell"from"the"larger"RNA"transcripts"that"are"transcribed"by"RNA"polymerase"II"(Lee"et"al.,"2004)."These"transcripts"(priXmiRNAs)"are"transcribed"by"a"complex"of"RNase"III"enzyme"along"with"Drosha,"

14""

and"the"doubleXstranded"RNAXbinding"protein,"DGCR8"(DiGeorge"syndrome"critical"region"gene"8)"to"form"small"stemXloop"(or"hairpin"shaped)"preXmiRNA"precursors"of"about"60X70"nucleotides"in"length"(Gregory"et"al.,"2004)."The"preXmiRNAs"are"then"actively"exported"by"exportinX5"to"the"cytoplasm,"where"they"lose"nucleotides"due"to"the"activity"of"the"cytoplasmic"RNase"III"enzyme"Dicer,"to"achieve"a"final"mature"miRNA"form"(Yi"et"al.,"2003)."To"exert"their"actions,"miRNA"must"selectively"attach"to"a"large"complex"of"proteins"called"RISC"(RNAXinduced"silencing"complex),"which"escort"miRNA"to"specific"mRNA"transcripts"for"their"final"destination"(Amarilyo"and"La"Cava,"2012)."At"this"point,"miRNA"can"diminish"the"production"of"proteins"through"two"major"mechanisms:"firstly,"the"attachment"of"antiXsense"sequences"to"specific"complementary"mRNA"targets"and"secondly"the"degradation"of"targeted"miRNAs."In"both"cases,"the"net"effect"of"miRNA"activities"is"the"repression"of"the"expression"of"targeted"gene"products"(Nilsen,"2007)."The"pathway"of"miRNA"is"shown"on"Figure,1.4.""Liston"et&al."reported"that"the"expression"of"miRs"are"firmly"controlled"during"haematopoiesis"and"lymphoid"cell"differentiation"(Liston"et"al.,"2010)."By"disabling"Dicer"or"RISC,"lineage"specific"disruption"of"the"entire"miR"network"leads"to"dysregulated"lineage"differentiation"and/or"activation,"thus"may"lead"to"autoimmunity.""Despite"the"vast"interest"in"miRNAs"expression"in"various"autoimmune"conditions,"there"have"not"been"comprehensively"studies"in"SS."A"study"conducted"by"Alevizos"et&al.,"investigated"miRNA"signatures"in"salivary"glands"(SG)"and"found"94"miRNAs"to"be"differentially"expressed"between"SS"patients"and"healthy"controls"(Alevizos"et"al.,"2011)."Kapsogeorgou"et&al."reported"that"there"is"overlapping"of"distinct"biologic"pathways"and"processes"in"SS"patients"with"miRNAs"associated"inflammation"or"SG"dysfunction"(Kapsogeorgou"et"al.,"2011)"suggesting"that"saliva"is"an"obvious"source"for"a"nonXinvasive"biomarker"in"Sjögren’s"syndrome"since"it"is"a"direct"product"of"the"affected"target"organ"(Alevizos"and"Illei,"2010)."Some"of"the"differentially"expressed"miRNAs"identified"by"researchers"(Iborra"et"al.,"2012;"Furer"et"al.,"2010)"may"have"diagnostic"and"prognostic"value"for"disease"type"and"severity"suggesting"that"miRNAs"are"

15""

implicated"in"the"pathogenesis"of"not"only"SS"but"also"autoimmune"diseases;"however"further"studies"are"still"required.""The"first"report"of"a"difference"in"miRNA"expression"between"SLE"patients"and"healthy"controls"dates"back"to"2007,"when"a"study"comparing"miRNA"expression"profiles"in"peripheral"blood"mononuclear"cell"(PBMC)"of"patients"with"SLE"and"idiopathic"thrombocytopaenic"purpura"(ITP)"patients,"identified"7"downregulated"and"9"upregulated"miRNA"in"SLE"as"compared"controls"(Dai"et"al.,"2007)."Preliminary"studies"have"shown"that"modulation"of"the"expression"of"dysregulated"miRNA"in"lupus"patients’"cells"can"influence"some"parameters"of"the"disease"(Amarilyo"and"La"Cava,"2012)."Yet"it"is"expected"that"factors"such"as"variability"of"symptoms,"organ"involvement"and"clinical"characteristics"of"the"SLE"patients"(due"to"genetic,"environmental"and"immunological"differences)"will"pose"challenges"to"translational"approaches"that"could"target"miRNA"in"SLE"for"therapeutic"benefits.""Due"to"the"complex"regulation"of"the"miRNA"networks"and"the"lack"of"inXvivo"functional"studies,"the"understanding"of"the"role"of"miRNA"regulatory"pathways"in"human"disease"remains"inadequate,"and"so"far"restricted"to"association"studies.""

1.10!Trim21!

Ro52"(or"Trim21)"is"a"common"target"of"circulating"autoantibodies"in"autoimmune"disease"(Oke"and"WahrenXHerlenius,"2012)."Trim21"contains"a"RING"and"a"BXbox"motif,"followed"by"a"coiledXcoil"(CC)"domain"and"a"B30.2"(or"PRYSPRY)"region"in"the"CXterminal"end"as"seen"in"Figure,1.5."

"

Figure!1.5:!The!structural!domains!of!Ro52.!Taken!from!(Oke!and!Wahren`Herlenius,!2012).!

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Autoantibodies"to"the"Ro52"protein"were"historically"denoted"by"the"common"name"antiXRo/SSA"antibodies."AntiXRo52"is"the"most"common"specificity"in"patients"with"pSS"(75.4%)"and"42.7%"in"SLE"patients"(SchulteXPelkum"et"al.,"2009)."However,"it"is"not"known"if"these"autoantibodies"affect"the"function"of"Ro52.""Bozic"et&al."have"reported"that"the"CC"domain"is"the"most"commonly"targeted"region"of"the"Ro52"protein"in"patients"with"SLE"(Bozic"et"al.,"1993)."Interestingly,"SS"patients"appear"to"have"specificities"against"several"different"Ro52"epitopes"including"the"Ring,"BXbox,"and"CC"domains.""Members"of"the"tripartite"motifXcontaining"protein"(TRIM)"family"are"single"protein"E3"ligases"(Higgs"et"al.,"2008)"that"have"ample"roles"in"cellular"biology."Ro52"(Trim21)"is"targeted"as"an"autoantigen"in"SLE"and"SS."The"RING"domains"of"TRIM"family"members"have"been"shown"to"have"E3"ubiquitin"ligase"activity"(Yoshimi"et"al.,"2012),"mediating"ubiquitination"events"such"as"protein"degradation,"trafficking,"and"activation."This"RING"domain,"according"to"Espinosa"et&al.,"is"required"for"the"E3"activity"of"Trim21"and"that"SS"and"SLE"patient"antiXRo52"autoantibodies"directed"to"the"RING"domain"inhibit"Ro52"activity"(Espinosa"et"al.,"2011)."They"concluded"that"antiXRo52"autoantibodies"might"have"indicated"a"direct"pathogenic"role"in"disease"development.""

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1.11!Aims!of!this!study!

The"study"is"divided"into"two"projects"with"the"first"half"looking"primarily"at"the"ability"of"the"drug"compounds"in"the"activity"of"Trim21"and"its"ability"to"reduce"type"I"interferon"leading"to"a"reduction"in"symptoms"and"causes"of"Systemic"Lupus"Erythematosus.""The"second"half"involves"research"on"microRNA"dysregulation"in"disease"process"in"Sjögren’s"Syndrome"that"could"ultimately"lead"to"aiding"in"the"development"of"highly"specific"and"personalised"microRNA"therapies.""The"main"aims"of"the"study"are"to:"

! Validate"drug"compounds"that"regulate"the"activity"of"Trim21"using"luciferase"reporter"assay."

! Apply"bioinformatics"research"and"analysis"to"identify"potential"microRNAs"whose"expression"is"dysregulated"in"pSS"patients"contributing"to"disease"onset"and/or"maintenance."

! Justify"miRs"of"interest"and"predicted"targets"on"PBMCs"of"pSS"patients"versus"healthy"controls"using"quantitative"polymerase"chain"reaction."

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2.0!Materials!and!Methods!

2.1!Materials!

"

2.1.1!General!Chemicals!and!Conditions!

All"chemicals"and"solutions"are"stored"at"room"temperature"20˚C"+/X"5˚C."All"solutions"were"prepared"using"pure"deionised"water.""Product! Storage! Supplier!

Coelentrazine" X20˚C" Argus"Fine"Chemicals"DMEM" " Biosera"Foetal"Bovine"Serum" X20˚C" Biosera"Phosphate"Buffered"Saline" " Biosera"Passive"lysis"buffer" 4˚C" Promega"Penicillin/"Streptomycin"Solution"100x" X20˚C" Biosera"PureYieldTM"Plasmid"MaxiPrep"System" " Promega"Trypan"Blue"0.5%"Solution" " Biosera"TrypsinXEDTA" 4˚C" Biosera"Gentamicin"50mg/ml"Solution" 4˚C" Sigma"Metafectene®" 4˚C" Biontex"Sodium"Hypochlorite"10X14%"RPMI"1640"FicollXPaque"Plus"Red"Blood"Cell"Lysis"Buffer"Taq"Polymerase"MyTaq™"Buffer"Tetro"cDNA"Synthesis"Kit"SensiFAST™"BCA"Protein"Assay"Kit"

"4˚C"""

X20˚C"X20˚C"X20˚C"X20˚C""

Lennox"Biosera"

GE"Healthcare"Sigma"Bioline"Bioline"Bioline"Bioline"

Thermo"Scientific""

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2.1.2!List!of!Suppliers!

Argus"Fine"Chemicals"Ltd,"Sussex"Innovation"Centre,"Science"Park"Square,"Falmer,"Brighton,"East"Sussex"BN1"9SB,"UK""Bioline"Reagents"Ltd,"Unit"16"The"Edge"Business"Centre,"Humber"Road,"London,"NW2"6EW,"UK""Biontex"Laboratories"GmbH,"Landsberger"Str."234,"80687"München,"Germany""Biosera,"1"Acorn"House,"The"Broyle,"Ringmer,"East"Sussex,"BN8"5NN,"UK""GE"Healthcare,"Pollards"Wood,"Nightingales"Ln,"Chalfont"St."Giles,"HP8"4SP,"UK""Lennox"Laboratory"Supplies"Ltd.,"John"F."Kennedy"Drive,"Naas"Road,"Dublin"12,"Ireland""Promega"Ltd.,"Delta"House,"Enterprise"Road,"Chilworth"Research"Centre,"Southhampton,"SO1"7NS,"UK""SigmaXAldrich"Company"Ltd.,"Fancy"Road,"Poole,"Dorset"BH12"4QH,"UK""Thermo"Fisher"Scientific"Inc.,"PO"BOX"117,"Rockford,"IL"61105,"USA""

2.2!Plasmid!Purification!

"

2.2.1!Transformation!of!Competent!Cells!with!Plasmid!DNA!

The"transformation"of"competent"cells"with"a"plasmid"DNA"molecule"is"a"technique"used"to"enable"the"plasmid"DNA"to"be"replicated"within"a"cell"independently"of"the"replication"of"the"cell’s"own"chromosome."The"technique"involves"the"addition"of"the"plasmid"DNA"to"the"competent"cells,"followed"by"the"plating"of"these"cells"in"bacterial"broth"onto"agar"plates"containing"an"antibiotic"that"the"plasmid"is"resistant"to."This"allows"for"the"growth"of"only"the"transformed"cells,"those"that"the"plasmid"has"been"acquired,"on"the"agar"plates.""

20""

2μl"of"respective"plasmid"DNA"was"added"to"20μl"aliquots"of"E.coli"competent"cells."The"contents"of"the"tube"were"mixed"gently"by"swirling"and"then"stored"on"ice"for"30mins."The"cells"were"heat"shocked"by"incubation"at"42˚C"for"45secs"and"then"cooled"on"ice"for"2mins."LB"broth"(200μl)"was"added"to"the"tube"and"the"cells"were"incubated"on"an"orbital"incubator"shaker"at"37˚C"for"1hr."This"allows"the"cells"to"recover."The"cells"were"then"plated"onto"LB"agar"plates"containing"1μl/ml"ampicillin,"and"incubated"at"37˚C"overnight.""Following"overnight"incubation,"bacterial"colonies"were"then"screened"for"the"required"insert"and"inoculated"onto"respective"flasks"with"100ml"LB"medium"containing"1μl/ml"penicillin"for"positive"selection"and"then"incubated"on"an"orbital"incubator"shaker"at"250rpm"overnight"at"37˚C.""

2.2.2!Maxiprep!from!Escherichia,coli!

Plasmid"purification"and"preparation"is"a"technique"used"to"extract"and"purify"the"plasmid"DNA"from"the"transformed"bacteria."Using"the"transformation"method"outlined"on"the"previous"page"and"the"purification"method"here,"a"stable"and"expressing"plasmid"is"easily"maintained"in"order"to"carry"out"overXexpression"studies"on"different"proteins.""Following"incubation,"cells"were"pelleted"at"3000rpm"for"30mins"at"4˚C."Plasmid"was"purified"using"Promega"PureYieldTM"Plasmid"Maxiprep"System."These"kits"were"used"according"to"the"manufacturer’s"instructions:"

o Cell"pellet"was"resuspended"in"12ml"of"Cell"Resuspension"Solution."o 12ml"of"Cell"Lysis"Solution"was"added"and"mixed"by"gentle"inversion"and"

the"incubated"at"room"temperature"for"3mins."o A"further"12ml"of"Neutralisation"Solution"was"added"to"the"lysed"cells"and"

mixed"by"inversion"10X15"times."Complete"precipitation"of"cellular"debris"must"occur"at"this"stage."

The"lysate"was"centrifuged"at"4000rpm"for"40mins"in"a"fixedXangle"rotor"at"room"temperature."When"finished,"the"lysate"should"be"clear,"with"small"amounts"of"cell"debris"remaining."

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2.2.3!DNA!Purification!

The"column"stack"was"assembled"by"placing"a"blue"PureYield™"Clearing"Column"on"top"of"a"white"PureYield™"

Maxi"Binding"Column"and"then"placed"onto"the"vacuum"manifold"(refer"to"Figure,2.1)."All"the"purification"and"elution"steps"were"performed"at"room"temperature.""

"

Figure! 2.1:! The! blue! PureYield™! Clearing!

Column! in! a!white! PureYieldTM!Maxi! Binding!

Column! on! a! Vac`Man®! Laboratory! Vacuum!

Manifold!port.!

The"respective"lysate"was"gradually"poured"onto"the"respective"blue"clearing"column"and"maximum"vacuum"force"was"applied."The"lysate"passed"through"the"clearing"membrane"in"the"clearing"column,"and"the"DNA"bound"to"the"binding"membrane"in"the"binding"column.""The"blue"clearing"column"was"removed,"only"leaving"the"white"binding"column"for"further"washing"steps."Endotoxin"

Removal"Wash"(5ml)"was"added"to"the"binding"column"and"with"the"aid"of"the"vacuum,"allowed"the"solution"to"be"pulled"through"the"column."Next"step,"involved"the"addition"of"20ml"of"Column"Wash"Solution"to"the"binding"column."Once"all"the"solution"has"wash"through,"the"membrane"was"dried"by"leaving"the"vacuum"applied"for"5mins"(refer"to"Figure,2.2).""

!

Figure!2.2:!Steps!in!Maxiprep!System!(Sigma`

Aldrich,!n.d.).!!

22""

2.2.4!Elution!of!DNA!by!Vacuum!

"The"elution"of"DNA"was"performed"using"the"Eluator™"Vacuum"Elution"Device"which"will"result"in"better"DNA"recovery"and"yield"compared"to"elution"by"centrifugation."A"sterile"1.5ml"microcentrifuge"tube"was"placed"on"the"base"of"the"elution"device,"securing"the"tube"cap"in"the"open"position,"as"shown"in"Figure,2.3."The"DNA"binding"column"was"then"inserted"on"top"of"the"device"and"placed"onto"the"vacuum"manifold."NucleaseXfree"water"(500μl)"was"added"to"the"binding"column"until"all"the"liquid"has"passed"through."The"microcentrifuge"tube"was"then"capped"and"saved"for"DNA"quantitation.""

"

Figure!2.3:!Assembly!of!microcentrifuge!tube!onto!the!base!of!the!Eluator™!Vacuum!Elution!Device.!

"

2.2.5!DNA!quantitation!using!the!NanoDrop!1000!Spectrophotometer!

The"NanoDrop"technique"was"used"to"accurately"determine"the"concentration"of"plasmid"DNAs"of"interest."A"volume"of"1μl"was"sufficient"for"accurate"measurements."1μl"of"nucleaseXfree"water"was"used"to"blank"the"assay"and"wiping"the"machine"after"each"addition"of"the"sample.

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2.3!Cell!Culture!

"

2.3.1!Growth!and!Maintenance!of!HEK293`T!cells!

Human"Embryonic"Kidney"(HEK)"293XT"cells"were"cultured"in"Dulbecco’s"Modified"Essential"Medium"(DMEM)"containing"stable"glutamine"with"sodium"pyruvate,"supplemented"with"10%"(v/v)"foetal"bovine"serum"(FBS),"2%"penicillinXstreptomycin,"and"50mg/ml"gentamicin."Cells"were"maintained"at"37˚C"in"a"humidified"atmosphere"of"5%"CO2."For"use"in"transfection"assays,"HEK293XT"cells"were"typically"seeded"at"1x105"cells/ml"24hr"prior"to"transfection."For"continuing"cell"culture,"cells"were"seeded"at"5x105"cells/ml"and"subXcultured"two"to"three"times"a"week."For"subXculture,"HEK293XT"cells"were"washed"in"5ml"of"Dulbecco’s"Phosphate"Buffered"Saline"(PBS)"and"then"incubated"at"27˚C"bathed"in"4ml"of"1x"Trypsin/"EDTA"(TE)"containing"0.05%"trypsin"and"0.02%"EDTA"for"5mins.""

2.3.2!Preparation!of!Instruments,!Culture!Medium,!and!Plates!

All"reagents"must"be"incubated"in"the"waterbath"at"37˚C"for"at"least"20mins."Turn"on"and"clean"the"laminar"flow"hood"with"70%"ethanol"in"order"to"work"under"sterile"conditions"at"all"times."Prepare"a"waste"beaker"filled"with"10X14%"sodium"hypochlorite"solution."To"maintain"complete"sterility,"all"instruments"and"solutions"going"into"hood"must"be"sprayed"with"ethanol."To"prepare"the"HEK293XT"cell"culture"medium,"aliquot"50ml"of"the"neat"medium"into"a"sterile"50ml"falcon"tube"and"label"as"serumXfree"DMEM."To"the"neat"bottle"of"medium,"add"50ml"of"FBS,"10ml"of"penicillinXstreptomycin"mixture"and"500μl"of"gentamycin.""After"neutralising"the"HEK293XT"cell"culture"flask,"10ml"of"DMEM"was"added"and"transferred"to"a"sterile"50ml"falcon"tube"and"centrifuged"at"1500"x"g"for"5mins"at"room"temperature."The"supernatant"is"aspirated"and"the"pellet"is"washed"with"2ml"of"DMEM,"depending"on"the"volume"of"the"pellet"(the"more"concentrated"the"pellet,"the"more"DMEM"was"added).""

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2.3.3!Cell!Counting!

Estimation"of"the"number"of"cells"present"in"the"cell"suspension"was"carried"out"using"a"haemocytometer"to"ensure"a"specific"number"of"cells"were"used"in"each"well"for"all"experiments."The"cell"pellet"was"resuspended"in"DMEM"and"a"10μl"sample"was"added"to"a"10μl"of"dye"(trypan"blue)."The"dye"is"used"to"assess"the"viability"of"the"cells,"as"it"is"expected"from"healthy"cells"but"is"takenXup"by"nonXviable"cells."10μl"of"the"mix"was"then"added"to"the"chamber"of"the"haemocytometer"with"the"number"of"cells"present"in"the"central"1mm2"grid"representing"0.1μl"of"the"cell"suspension."The"number"of"cells/ml"was"calculated"(cells/μl"x"dilution"factor"x"10,000)"and"from"this"the"required"volume"of"cell"suspension"was"seeded"for"each"well"in"a"96Xwell"plate."Each"well"was"seeded"with"200μl"of"cell"suspension.""

2.3.4!Sub`culture!of!HEK293`T!cells!

From"the"cell"suspension,"1ml"of"the"cell"suspension"was"added"to"the"TX75"cell"culture"flask"with"34ml"of"DMEM"and"then"incubated"at"37˚C.""

2.3.5!Transient!Transfection!

When"cells"are"transiently"transfected"with"plasmids,"the"DNA"is"introduced"into"the"nucleus"of"the"cell,"but"does"not"integrate"into"the"chromosome."This"means"that"many"copies"of"the"gene"of"interest"are"present,"leading"to"high"levels"of"expressed"protein."""Metafectene®"is"a"liposomal"based"transfection"reagent"from"Biontex"and"was"used"for"transfection"of"HEK293XT"cells."For"96Xwell"plate"transfections,"cells"were"seeded"at"1x105"cells/ml"and"grown"overnight."Cells"were"transfected"in"triplicates"per"transfection."The"volume,"per"plate,"was"brought"up"to"1050μl"with"serumXfree"DMEM."86.1μl"Metafectene"was"mixed"with"963.9μl"serumXfree"DMEM"per"transfection"of"plate"and"incubated"at"room"temperature"for"5mins."30μl"of"this"mixture"was"added"to"triplicate"amounts"of"100ng"DNA"(1.75μl"of"P125,"Renilla,"TRIF,"TRIM21,"and"EV"per"respective"well)"and"incubated"for"15mins"at"room"temperature."DNA/"Metafectene/"serumXfree"DMEM"

25""

(transfection"mix)"was"then"added"to"the"cells"(10μl"per"well"–"refer"to"Figure,2.4),"which"were"allowed"to"recover"for"24hr"at"37˚C"prior"to"cell"lysis."Cells"for"all"experiments"were"harvested"18hr"postXtransfection.""

"

Figure!2.4:!Layout!of!a!96`well!plate!transfection!for!the!addition!of!plasmid!DNAs.!

"

2.3.6!Addition!of!Compounds!

Following"18hr"transfection,"compounds"of"interest"are"added"to"the"transfected"96Xwell"plate."Depending"on"the"dose"response"concentrations,"the"volume"of"compounds"added"to"the"wells"varies."For"5μM"concentration,"10μl"of"the"stock"compound"was"added"directly"to"the"respective"wells"in"triplicates;"500nM"concentration,"1μl"of"the"stock"compound"was"also"added"directly"to"the"corresponding"wells"in"triplicates."For"50nM"concentration,"2μl"of"the"stock"was"diluted"to"18ul"of"DMEM"and"1μl"of"this"mix"was"added"to"the"respective"wells"in"triplicates."Finally,"5nM"concentration,"2μl"of"the"50nM"solution"was"diluted"to"18μl"of"DMEM"and"1μl"of"this"mix"was"added"to"the"corresponding"wells"in"triplicates."The"plate"is"then"incubated"at"37˚C"for"6hr.""

26##

2.4$Luciferase$Assay$

Luciferase#reporter#assays#are#a#useful#method#for#studying#gene#expression#

downstream#of#a#specific#enhancer#of#downstream#signalling.#The#luciferase#

reporter#assay#uses#two#reporter#enzymes,#Firefly#and#Renilla#(refer#to#Figure'

2.5).#Both#of#these#enzymes#are#situated#within#specific#light#emitting#plasmids#

that#give#off#light#when#stimulated.#The#amount#of#light#given#off#directly#

correlates#to#the#activity#of#the#system#being#studied.#The#Renilla#enzyme#acts#as#

an#internal#control#to#serve#as#a#baseline#response#and#the#Firefly#enzyme#

correlates#to#the#effect#of#the#specific#experimental#conditions.#By#normalising#

activity#of#the#Firefly#enzyme#to#that#of#the#Renilla#enzyme#minimises#

experimental#variability#caused#by#cell#viability#or#transfection#efficiency.#

#

Figure$2.5:$Dual$reporter$luciferase$assay$system.$

#

HEK293IT#cells#were#transfected#in#96Iwell#plates#as#described#above.#Following#

18hr#transfection,#the#media#was#removed#from#HEK293IT#cells#and#the#cells#

were#lysed#for#at#least#20mins#on#a#rocking#platform#at#room#temperature#with#

50μl#1x#Passive#Lysis#Buffer#(Promega).#Firefly#luciferase#activity#was#

27

determined using 20μl of cell lysate and an equivalent amount (20μl) was used

for determination of Renilla luciferase activity.

Firefly luciferase activity was assayed by the addition of 40μl of luciferase assay

mix (20mM tricine, 1.07mM (MgCO3)4Mg(OH)2.5H2O, 2.6mM MgSO4, 0.1mM

EDTA, 33.3mM DTT, 270mM coenzyme A, 470mM luciferin, 530mM ATP) to the

sample and Renilla luciferase activity was assayed by the addition of 40μl of a

1:1000 dilution of Coelentrazine in PBS. Luminescence was read using Wallac

luminometer. Firefly luminescence readings were corrected for Renilla activity

and expressed as fold stimulation over unstimulated empty vector (EV) control.

2.5 Bicinchoninic Acid (BCA) Assay

The BCA assay is a biochemical assay commonly performed to determine total

concentration of protein in a solution. It relies primarily on two reactions, as

shown in Figure 2.6.

Figure 2.6: Schematic diagram of the reaction for the bicinchoninic acid-‐containing protein assay

(White, 2007).

The concentration of the protein needed for the thermal melt shift assay was

conducted using the BCA method.

28""

Table!2.1:!Procedure!for!making!up!the!BCA!Standards!as!per!manufacturer’s!guidelines.!

""25μl"of"each"standard"was"pipetted"in"triplicates"on"a"96Xwell"plate."A"working"reagent"composed"of"50:1"diluted"ratio"of"Reagent"A:"Reagent"B"was"made"and"200μl"was"added"to"each"well."Equivalent"amounts"applied"to"the"protein"samples"as"well."The"plate"was"then"placed"on"a"rocking"platform"at"room"temperature"for"1min"and"incubated"at"37˚C"for"30mins."Following"incubation,"the"plate"was"read"on"a"Wallac"Victor3"V"multiplate"reader"at"570nm.""

2.6!Thermal!Melt!

A"thermal"shift"assay,"also"called"differential"scanning"fluorimetry"(DSF),"is"a"thermal"denaturation"assay"which"measures"the"thermal"stability"of"a"target"protein"and"a"subsequent"increase"in"protein"melting"temperature"upon"binding"of"a"ligand"to"the"protein."The"binding"of"low"molecular"weight"ligands"can"increase"the"thermal"stability"of"a"protein."The"thermal"stability"change"is"measured"by"performing"a"thermal"denaturation"curve"in"the"presence"of"a"fluorescent"dye,"such"as"Sypro"Orange."Sypro"orange"binds"nonXspecifically"to"hydrophobic"surfaces,"and"water"strongly"quenches"the"fluorescence"of"Sypro"orange"(Kranz"and"SchalkXHihi,"2011)."When"the"protein"unfolds,"the"exposed"hydrophobic"surfaces"bind"the"dye"resulting"in"an"increase"in"fluorescence"(refer"to"Figure,2.7)."The"shifting"of"the"curve"to"the"left"or"right"of"the"reference"decides"the"binding"of"the"ligands."""

29""

Before"doing"the"thermal"melt"assay,"it"is"important"to"determine"the"concentration"of"the"protein"to"use"as"well"as"the"concentration"of"the"solubilising"agent,"which"in"this"case"is"dimethyl"sulphoxide"(DMSO)."This"was"established"by"doing"a"dose"response"concentration"of"the"protein"and"DMSO"and"running"it"on"an"Applied"Biosystems"7900HT"Fast"RealXTime"PCR"machine"qPCR"machine.""To"avoid"extensive"protein"degradation,"it"was"necessary"to"place"the"protein"sample"to"an"ice"bucket.""Once"the"protein"and"DMSO"concentrations"have"been"determined,"the"drug"compounds"were"then"tested"for"their"thermal"stability."Each"drug"compound"was"made"up"with"5μl"of"50x"Sypro"orange"dye,"4.4μl"of"protein,"2.5μl"of"the"100μM"stock"drug"compound,"and"13.1μl"of"trisXbuffered"saline"(TBS)"buffer"to"a"final"volume"of"25μl."From"this,"20μl"was"aspirated"and"placed"on"a"96Xwell"plate"and"run"on"an"Applied"Biosystems"7900HT"Fast"RealXTime"PCR"machine.""

"

Figure!2.7:!Schematic!diagram!for!high!throughput!dynamic!scanning!fluorimetry!thermal!stability!

assay.!A,!illustrates!the!sample!preparation!and!B,!represents!the!native,!denatured,!and!aggregated!

states!of!protein!alongside!its!typical!exogenous!fluorescence!response!(Vollrath!et!al.,!2014).!

30""

2.7!Bioinformatics!Analysis!of!miR!Screen!Data!

A"miR"screen"was"conducted"to"determine"the"expression"profile"of"miRs"in"peripheral"blood"mononuclear"cells"(PBMCs)"from"SLE"and"pSS"patients"and"healthy"controls"to"identify"potential"miRs"that"may"be"contributing"to"disease"onset"and"maintenance."From"the"Nanostring"miR"screen"data"of"~800miRs,"potential"miRs"of"interest"were"selected"for"further"investigation."miRs"with"an"expression"ratio"above"2Xfold"(upregulated)"and"below"0.5Xfold"(downregulated)"were"chosen"based"on"Nanostring’s"recommended"guidelines.""A"table"was"constructed"under"the"following"headings"in"relation"to"each"miR"found:"miRs" "" miRs"of"interest""Predicted"targets" "" Predicted"gene"target"Gene"description" "" The"function"of"the"predicted"gene"target."Number"of"binding"sites"(BS)" "" The"number"of"binding"sites"the"miR"has."Target"score"(TS)" "" How"likely"a"predicted"gene"target"is"to"be"real."

If"the"predicted"target"score"is">80"then"it"is"most"likely"real"and"<60"is"not"recommended."

Other"databases"(DB)" "" The"number"of"other"databases"the"predicted"gene"target"has"been"associated"with"the"miR."

Role"in"immune"process" "" The"immune"role"the"predicted"gene"target"has"been"associated"with."

Disease"association" "" The"type"of"disorders"the"miR"is"associated"with"in"addition"to"autoimmune"disorders."

Reference" "" Reference"the"publication"found.""Based"on"the"findings,"potential"genes"of"interest"were"selected."

31""

2.8!Primer!Design!and!Optimisation!

The"forward"and"reverse"primers"of"the"miRs"of"interest"as"well"as"the"gene"targets"were"identified"using"PCR"primer"design"programs."The"miRs"of"interest"were"inputted"into"a"microRNA"database,"miRBase"(http://www.mirbase.org)."“Get&stemDloop&sequence”"was"selected,"revealing"the"sequence."All"the"uracil"nucleotides"were"replaced"with"thymine"bases."A"region"of"~20"base"pairs"near"the"5’"sequence"was"taken"as"the"forward"primer"and"a"region"of"~20"base"pairs"at"the"3’"end"sequence"was"taken"as"the"reverse"primer."As"the"DNA"is"transcribed"in"a"5’"""3’"direction,"it"was"necessary"for"the"reverse"primer"to"be"transformed"into"its"reverse"complement"counterpart"using"http://reverseXcomplement.com.""The"specificity"of"the"primers"was"checked"using"UCSC’s"Genome"Bioinformatics"(https://genome.ucsc.edu)."Under"tools,"“InDsilico&PCR”"was"selected,"which"searches"a"sequence"database"with"a"pair"of"PCR"primers"(Forward"and"Reverse),"using"an"indexing"strategy."The"primer"sequences"were"given"and"listed.""The"gene"targets"were"logged"onto"Harvard’s"PrimerBank"and"the"highest"amplicon"size"was"selected"and"its"respective"forward"and"reverse"primer"sequences,"length,"and"annealing"temperature"(Tm)"were"listed.""The"primers"were"then"optimised"to"determine"its"best"annealing"temperature"to"work"with."The"primers"were"diluted"according"to"the"manufacturer’s"guidelines"with"nucleaseXfree"water"under"the"PCR"hood"to"ensure"complete"sterility."From"this,"a"20μM"stock"solution"was"made"(50μl"of"primers"diluted"with"200μl"of"nucleaseXfree"water)."A"master"mix"was"then"prepared"with"the"following"components:"

" 1"Reaction" 3"Reactions"MyTaq"Buffer" 5μl" 16.5μl"Forward"Primer" 1μl" 3.3μl"Reverse"Primer" 1μl" 3.3μl"Taq"Polymerase" 0.125μl" 0.41μl"NucleaseXfree"water" 15.375μl" 50.74μl"

"

32""

From"the"master"mix,"22.5μl"was"aspirated"and"added"to"200μl"PCR"tubes"with"2.5μl"cDNA."These"tubes"were"then"placed"on"the"ProFlex"PCR"System"to"be"optimised"with"the"following"conditions:"

PCR"cycling"conditions" Temp."/"Time"points"Initial"denaturation" 2mins"@"95˚C"Denaturation" 30secs"@"95˚C"Annealing" 30secs"@"55˚C"Extension" 30secs"@"72˚C"Final"extension" 10mins"@"72˚C"

"

2.9!Gel!Electrophoresis!(1%!Agarose)!

Gel"electrophoresis"is"a"laboratory"method"typically"used"to"separate"DNA,"RNA,"or"proteins"according"to"their"molecular"size."The"molecules"to"be"separated"are"pulsed"by"an"electric"field"through"a"gel"containing"small"pores."The"molecules"travel"though"the"pores"in"the"gel"at"a"speed"that"is"inversely"related"to"their"lengths"meaning"a"small"DNA"molecule"tends"to"travel"at"a"greater"distance"through"the"gel"than"a"larger"DNA"molecule.""

"

Figure!2.8:!Schematic!diagram!of!the!steps!involved!in!gel!electrophoresis!(PremedHQ,!2001).!

33""

A"1%"Agarose"gel"contains"0.35g"of"agarose"diluted"into"35ml"of"1x"trisXacetateXEDTA"(TAE)"buffer."The"agarose"was"then"heated"in"the"microwave"for"approximately"2mins"at"800Watts"with"occasional"swirling."Once"all"the"crystals"have"dissolved,"3.5μl"of"SyberSafe"DNA"gel"stain"was"added"to"the"mixture"and"swirled"to"ensure"complete"homogeneity."The"solution"was"allowed"to"cool"to"approximately"55˚C"before"pouring"into"the"casting"tray"and"allowed"to"solidify"for"30mins."Once"the"gel"has"hardened,"the"comb"was"gently"removed"and"the"gel"was"completely"flooded"with"TAE"buffer"(up"to"2X3mm"above"the"gel).""5μl"of"molecular"DNA"ladder"was"pipetted"onto"the"first"well"of"the"gel"and"15μl"of"samples"into"the"subsequent"wells."The"electrode"wires"were"connected"to"the"power"supply"at"one"end"and"to"the"rig"apparatus"at"the"other"ensuring"the"black"cable"was"connected"to"the"negative"electrode"and"the"red"cable"to"the"positive"electrode."An"electric"field"of"120V"for"30mins"was"applied"and"the"current"checked"by"the"presence"of"bubbles"at"each"electrode"(refer"to"Figure,2.8).""Following"separation"of"the"DNA,"the"gel"was"visualised"under"UV"light"using"the"G:Box"imaging"system"and"the"molecular"weight"of"the"PCR"product"determined"by"comparing"it"to"the"DNA"molecular"weight"ladder.""

2.10!Isolation!of!Peripheral!Blood!Mononucleated!Cells!(PBMCs)!

10%"sodium"citrate"anticoagulated"blood"was"diluted"1:1"with"Dulbecco’s"PBS"and"completely"layered"on"an"identical"volume"of"the"density"gradient,"which"contained"FicollXPaque"Plus"reagent."Blood"samples"were"centrifuged"for"30mins"at"1000"x"g&and"20˚C"without"applying"a"brake."The"PBMC"interface"was"carefully"removed"by"pipetting"and"the"buffy"coat"layer"was"aspirated"using"a"sterile"Pasteur"pipette"onto"a"new"falcon"tube"and"the"volume"was"adjusted"to"50ml"with"PBS."The"tubes"were"centrifuged"for"5mins"at"1500"x"g"with"the"highest"brake"setting."The"pellet"was"resuspended"in"5ml"Red"Cell"Lysis"buffer"in"the"presence"of"red"blood"cells"and"the"volume"adjusted"to"50ml"with"PBS;"centrifuged"for"5mins"at"1500"x"g."PBMC"were"resuspended"in"5ml"RPMI"medium"and"an"appropriate"volume"of"TRIzol"reagent"was"added"at"stored"at"X80˚C."

34

2.11 Isolation and Quantitation of mRNA

Following isolation of PBMC, the suspension cells was homogenised and

incubated for 5mins at RT. 200µl of chloroform was added per ml of TRIzol

reagent used and shook vigorously for 15secs and allowed to stand for 15mins at

RT; followed by centrifugation at 12,000 x g for 15mins and 4˚C to allow the

separation of the mixture into three distinct phases: a red organic phase (protein

content), DNA containing interphase, and a colourless upper aqueous phase

which contains RNA. The upper aqueous phase was transferred onto a new

eppendorf tube and 500µl of 2-‐propanol added per ml of TRI reagent used and

allowed to stand for 30mins at RT. The tubes were centrifuged at 12,000 x g for

10mins at 4˚C. The RNA pellet was washed with 1ml 75% ethanol, vortexed and

centrifuged at 12,000 x g for 10mins at 4˚C. Following recentrifugation, the

supernatant was aspirated under a vacuum and the pellet was resuspended in

30µl nuclease-‐free water. mRNA was then quantified using the NanoDrop

spectrophotometer.

2.12 Synthesis of cDNA

Subsequent to mRNA isolation, the required amount of mRNA stock was

calculated to attain the same concentration of mRNA in all samples designed to

convert up to 5µg of total RNA (diluted in nuclease-‐free water). Tetro cDNA

Synthesis Kit (Bioline, London, UK) was used as a reverse transcription system to

generate cDNA from an RNA template:

Volume 5x RT Buffer 4µl Random Hexamer Primer Mix 1μl dNTP Mix 1μl RNase Inhibitor 1μl Reverse Transcriptase 1μl

8μl aliquots of the reaction mix were added to 12μl of mRNA sample for a final

volume of 20μl and mixed gently by pipetting. The tubes were then placed on the

DNA Engine® Peltier Thermal Cycler PTC-‐200 (MJ Research) PCR machine with

the following cycles and cDNA samples were then quantified using the NanoDrop

spectrophotometer:

35""

10mins"@"25˚C"30mins"@"45˚C"5mins"@"85˚C"∞"@"4˚C"

"

2.13!Quantitative!PCR!(qPCR)!

In"quantitative"or"realXtime"PCR,"amplification"of"DNA"is"monitored"at"each"cycle"of"the"reaction."When"the"DNA"is"in"the"log"linear"phase"of"amplification,"the"amount"of"fluorescence"increases"above"the"background,"as"seen"in"Figure,2.9.""

"

Figure!2.9:!Real`time!PCR!mechanism!(Sigma`Aldrich,!2002).!

A"master"mix"of"the"following"components"was"prepared:"" 1"Reaction"2x"SensiFAST" 10μl"Forward"Primer" 1μl"Reverse"Primer" 1μl"NucleaseXfree"water" 7μl"

"19μl"of"the"master"mix"was"added"to"1μl"of"cDNA"in"each"well"for"a"final"volume"of"20μl/well"of"a"96Xwell"plate."The"plate"was"sealed"with"a"flat"8Xcap"strip"and"spun"down"at"room"temperature"for"10secs"to"remove"air"bubbles."The"plate"was"placed"into"the"Applied"Biosystems"7500"RealXTime"PCR"machine"and"the"following"conditions"was"setXup:"

Cycling"conditions" Temp."/"Time"points"Initial"denaturation" 2mins"@"95˚C"Denaturation" 15secs"@"95˚C"Annealing" 15secs"@"55˚C"Extension" 45secs"@"72˚C"

36""

3.0!Results!

3.1!Plasmid!DNA!Purification!

The"following"are"the"concentrations"of"plasmid"DNA"required"for"transfection"on"luciferase"assays"(Table,3.1)."The"concentrations"were"determined"using"the"NanoDrop"technique."These"plasmid"DNAs"are"then"stored"as"stock"solutions"and"100ng"of"each"are"prepared"for"transfection,"as"detailed"in"Section"2.3.5.""

Table!3.1:!Concentrations!of!Plasmid!DNA!determined!by!NanoDrop.!

DNA"Plasmid" 1st"Reading" 2nd"Reading" 3rd"Reading" Mean"Reading"EV" 249.7ng/μl" 244.9ng/μl" 242ng/μl" 245.5ng/μl"P125" 190.4ng/μl" 200ng/μl" 195.3ng/μl" 195.2ng/μl"Renilla" 215.1ng/μl" 230.5ng/μl" 212.7ng/μl" 219.4ng/μl"TRIF" 40.3ng/μl" 46.5ng/μl" 48.6ng/μl" 45.1ng/μl"TRIM21" 390.5ng/μl" 389.4ng/μl" 404.2ng/μl" 394.7ng/μl""Luciferase"reporter"assays"were"employed"to"ascertain"what"effect,"if"any,"the"compounds,"3A"and"4A,"had"on"the"activity"of"Trim21"as"an"inhibitor"of"TRIFXdriven"IFNXβ"promoter"activity."Both"3A"and"4A"compounds"have"been"previously"assessed"in"the"lab"and"had"shown"to"have"promising"results."HEK293XT"cells"were"seeded"at"1x105"cells/ml"and"transfected"with"plasmids"containing"TRIF"(100ng),"to"drive"the"system,"P125"(100ng),"Renilla"luciferase"(100ng),"and"EV"(100ng),"together"with"Trim21"(100ng)."18"hours"postXtransfection"the"cells"were"treated"with"increasing"concentrations"(0.005μM,"0.05μM,"0.2μM,"0.5μM,"1μM,"5μM,"25μM)"of"relevant"compounds"for"6"hours."Following"treatment"of"the"cells"with"the"relevant"small"compounds,"cells"were"lysed"and"the"luciferase"reporter"assay"was"carried"out"in"order"to"determine"the"amount"of"IFNXβ"promoter"activity"in"the"cell.""Initially,"the"project"started"with"testing"the"individual"components"of"our"vehicle"controls"(VC)"to"which"the"compounds"were"solubilised"in."Figure,3.1"shows"the"data"for"each"of"the"VC"constituents."We"can"see"that"TRIF"drives"IFNXβ"promoter"activity,"which"is"acting"as"a"positive"control."The"system"requires"a"negative"regulator"to"balance"the"system."Trim21"has"been"characterised"to"negatively"

37""

regulate"TRIFXdriven"IFNXβ"promoter"activity."The"addition"of"Trim21"to"the"system"inhibits"TRIF"driven"IFNXβ"promoter"with"a"52.56%"inhibition"(refer"to"Table,3.2)."Therefore,"TRIF+TRIM21"is"our"threshold"level"to"which"we"expect"our"vehicle"controls"to"be"at"the"same"or"similar"level"and"our"drug"compounds"to"be"significantly"less"than"our"threshold"level."We"expect"the"compounds"to"be"less"suggesting"that"the"small"molecules"are"driving"the"inhibition"of"the"system."A"VC"relative"to"each"concentration"of"the"compound"was"employed"since"the"drug"compounds"were"solubilised"in"properties"other"than"water,"such"as"cyclodextrin"(CD),"polyethylene"glycol"(PEG),"and"sodium"hydroxide"(NaOH)."""Based"on"previous"results"from"the"lab,"we"decided"to"utilise"a"single"concentration"of"the"drug"compounds"at"0.05μM"with"different"diluents"including"VC"itself,"PBS"and"sterile"filtered"DMEM."Unfortunately,"although"3A"PEG"compound"looks"to"be"inhibiting"IFNXβ"promoter"activity"(refer"to"Figure,3.2)"as"its"respective"VC"are"that"almost"equal"to"the"level"of"TRIF+TRIM21,"the"error"bars"associated"proved"to"be"a"significant"indication"that"these"results"could"not"be"validated."Further"tests"and"analysis"of"the"cell"line"resulted"in"conclusion"that"it"was"mycoplasma"infection."Mycoplasma"can"induce"a"myriad"of"cellular"changes"that"can"lead"to"a"false"interpretation"of"our"experimental"data"3A"CD"compound"on"the"other"hand"does"not"show"indicate"inhibition"of"the"drug"compounds."As"with"4A"CD"and"4A"PEG"compounds"(refer"to"Figure,3.3),"the"same"can"be"said"in"that"validation"of"results"could"not"be"justified"due"to"the"high"levels"of"the"error"bars.,"Upon"further"assessment"of"the"results,"we"have"decided"to"return"to"conducting"a"dose"response"rather"than"applying"just"a"single"concentration"mainly"due"to"no"consistency"with"the"expected"results"where"the"VC"should"be"at"a"level"proportionate"to"TRIF+TRIM21"and"the"drug"compounds"inhibiting"the"system."

38""

3.2!Luciferase!Assay!

"

"

Figure!3.1:!Trim21!negatively! regulates!TRIF!driven! IFN`β!promoter! activity.!HEK293T`cells!were!

seeded! at! 1x105! cells/ml,! 200μl/well,! in! a! 96`well! plate.! Cells! were! transfected! with! plasmids!

containing!100ng!of!TRIF!to!drive!the!system,!P125,!Renilla,!Trim21,!and!an!EV!control.!18hr!post`

transfection! cells! were! harvested! and! compounds! added! and! luciferase! assay! was! measured!

following!6hr!incubation.!A,!cyclodextrin!(CD)!vehicle!control;!B,!polyethylene!glycol!(PEG)!vehicle!

control;! and! C,! sodium! hydroxide! (NaOH)! vehicle! control.! The! above! data! is! graphed! from! the!

average!of!three!separate!experiments,!±!standard!error!of!the!mean!SEM!(n=3).!

0

50

100

150Fold(expression

CD Vehicle'ControlControls

0

50

100

150

Fold(expression

PEG

EV

TRIF

TRIM21

TRIF+TRIM21 50 5

0.5

0.05

0

50

100

150

200

Drug2Concentration2(μM)

Fold2expression

NaOH

A

B

C

39""

"

"





following! 6hr! incubation.! A,! 3A, compound! in! cyclodextrin! (CD)! vehicle! control! with! diluents!

including!VC!itself,!PBS,!and!sterile`filtered!DMEM;!and!B,!3A,compound!in!polyethylene!glycol!(PEG)!

vehicle! control!with! diluents! including!VC! itself,! PBS,! and! sterile`filtered!DMEM.!Both! compounds!

are! at! 0.05μM! concentration.! The! above! data! is! graphed! from! the! average! of! three! separate!

experiments,!±!SEM!(n=3).!

A

B

0

20

40

60

80

100

120

Fold+expression

3A+CD Vehicle'ControlDrug'CompoundControls

EV

TRIF

TRIM21

TRIF+TRIM21 VC

PBS

MEDIA

0

20

40

60

80

100

120

Diluents

Fold>expression

3A>PEG

40""

"

"





following! 6hr! incubation.! A,! 4A, compound! in! cyclodextrin! (CD)! vehicle! control! with! diluents!

including!VC!itself,!PBS,!and!sterile`filtered!DMEM;!and!B,!4A,compound,in!polyethylene!glycol!(PEG)!

vehicle! control!with! diluents! including!VC! itself,! PBS,! and! sterile`filtered!DMEM.!Both! compounds!

are! at! 0.05μM! concentration.! The! above! data! is! graphed! from! the! average! of! three! separate!

experiments,!±!SEM!(n=3).!!

A

B

0

20

40

60

80

100

120

Fold+expression

4A+CD Vehicle'ControlDrug'CompoundControls

EV

TRIF

TRIM21

TRIF+TRIM21 VC

PBS

MEDIA

0

20

40

60

80

100

120

Diluents

Fold>expression

4A>PEG

41""

A"new"batch"of"HEK293XT"cells"were"seeded"at"1x105"cells/ml"and"transfected"with"100ng"of"plasmids"containing"TRIF,"P125,"Renilla,"EV,"and"Trim21."18"hours"postXtransfection"the"cells"were"treated"with"increasing"concentrations"(0.005μM,"0.05μM,"0.5μM,"and"5μM)"of"relevant"compounds"for"6"hours."Following"treatment,"the"cells"were"lysed"and"the"luciferase"reporter"assay"was"carried"out."Figure,3.4"shows"the"treatment"of"transfected"HEK293XT"cells"with"3A"compounds"in"CD,"PEG,"and"TFA."None"of"the"compounds"made"any"difference"to"its"effects"on"the"IFNXβ"promoter"activity"with"the"respective"VCs"much"higher"than"the"threshold"level"of"TRIF+TRIM21,"although"inhibition"of"TRIF"driven"IFNXβ"promoter"activity"by"Trim21"is"quite"significant"(p<0.05)"with"33.1%"inhibition"(refer"to"Table,3.2)."At"the"lowest"concentration"of"3A"TFA"(Figure,3.4C),"the"vehicle"control"was"quite"significant"(p<0.05)"using"the"Bonferroni"correction"on"GraphPad"Prism"6"(v6.0f)"with"a"value"of"p=0.0222.""The"treatment"of"transfected"HEK293XT"cells"with"4A"compounds"in"CD,"PEG,"and"TFA"can"be"seen"in"Figure,3.5."This"compound"seems"to"be"the"only"one"to"show"a"significant"increase"in"the"inhibition"of"TRIF"driven"IFNXβ"promoter"activity"(p<0.05)."Figure,3.5A"has"shown"more"statistically"significant"results"of"the"drug"compound"but"unfortunately"its"respective"VCs"looks"to"be"inhibiting"IFNXβ"promoter"activity"as"well,"which"is"not"what"we"would"expect."On"the"other"hand,"Figure,3.5B"looks"to"be"promising"with"the"VCs"almost"proportionate"to"the"TRIF+TRIM21"threshold"and"the"PEG"compounds"can"be"viewed"to"be"inhibiting"IFNXβ"promoter"activity."However,"they"have"not"been"seen"to"be"statistically"significant."At"the"top"concentration"of"5μM,"4A"TFA"(refer"to"Figure,3.5C)"is"very"encouraging"in"that"its"VC"is"similarly"equal"to"the"threshold"level"and"its"drug"is"significantly"less"(p<0.05)."This"suggests"that"4A"compounds"show"a"great"selectivity"for"Trim21"than"compared"to"3A"compounds.

42""

"





following!6hr! incubation.!A,!3A,compound! in! cyclodextrin! (CD)! vehicle! control;!B,!3A,compound,in!

polyethylene!glycol!(PEG)!vehicle!control;!and!C,!3A,compound! in!trifluoroacetic!acid!(TFA)!vehicle!

control.!The!above!data! is! graphed! from! the!average!of! three! separate!experiments,!±! SEM! (n=3).!

*p<0.05!was!considered!significant.!

A

B

C

0

5

10

15

20

25

Fold)expression

3A)CD Vehicle'ControlDrug'CompoundControls

0

5

10

15

20

25

Fold)expression

3A)PEG

EV

TRIF

TRIM21

TRIF+TRIM21 5

0.5

0.05

0.0050

5

10

15

20

25


Fold2expression

3A2TFA*

43""

"





following!6hr! incubation.!A,!4A,compound! in! cyclodextrin! (CD)!vehicle! control;!B,!4A!compound! in!

polyethylene!glycol!(PEG)!vehicle!control;!and!C,!4A,compound,in!trifluoroacetic!acid!(TFA)!vehicle!



A

B

C

0

50

100

150

200

250

Fold)expression

4A)PEG0

50

100

150

200

250

Fold)expression

4A)CD Vehicle'ControlDrug'CompoundControls

**

**

*

EV

TRIF

TRIM21

TRIF+TRIM21 5

0.5

0.05

0.0050

50

100

150

200

250


Fold2expression

4A2TFA

*

44""

With"promising"results"for"the"4A"compounds,"we"have"decided"to"treat"the"transfected"HEK293XT"cells"with"increasing"concentrations"of"the"compounds,"including"3A,"to"0.2μM,"1μM,"5μM,"and"25μM"doses."HEK293XT"cells"were"seeded"at"1x105"cells/ml"and"transfected"with"100ng"of"plasmids"containing"TRIF,"P125,"Renilla,"EV,"and"Trim21."18"hours"postXtransfection"the"cells"were"then"treated"the"relevant"compounds"for"6"hours."Following"treatment,"the"cells"were"lysed"and"the"luciferase"reporter"assay"was"carried"out.""Previously,"we"were"using"miniscule"volumes"of"treatment"of"the"drugs"that"perhaps"pipetting"error"could"not"easily"be"dismissed."With"this,"we"decided"to"treat"the"transfected"cells"with"an"equal"volume"of"50μl"of"the"respective"drug"compounds."This"way,"we"would"only"use"one"VC"for"all"the"concentration"of"the"drugs."However,"a"miscalculation"on"my"behalf"resulted"in"the"errors"with"the"VC."We"needed"a"different"VC"for"the"respective"concentration"of"the"compound"due"to"the"difference"in"volumes"of"stocks"made"up."Therefore,"as"clearly"seen"in"Figure,3.6,and"Figure,3.7,"only"one"VC"for"the"top"concentration"of"25μM"was"used."Unfortunately,"despite"the"experimental"flow,"it"is"also"very"clear"that,"perhaps,"Trim21"does"not"look"like"its"inhibiting"IFNXβ"promoter"activity"or"that"TRIF"is"not"promoting"the"activity"of"IFNXβ."Therefore,"this"tells"us"that"the"results"could"not"be"validated.""By"this"stage,"we"had"another"look"at"our"cells"and"we"concluded"that"it"was"again"infected,"possibly"with"mycoplasma."Due"to"this,"our"experimental"results"cannot"be"accurately"interpreted.""The"percentage"inhibition"for"each"of"the"respective"VC"and"drug"compounds"(DC)"have"also"been"calculated"and"listed"in"Table,3.2"and"TRIF"acting"as"our"reference"with"0%"inhibition."

45""

"





following!6hr! incubation.!A,!3A,compound! in! cyclodextrin! (CD)! vehicle! control;!B,!3A,compound,in!

polyethylene!glycol!(PEG)!vehicle!control;!and!C,!3A,compound,in!trifluoroacetic!acid!(TFA)!vehicle!



A

B

C

0

20

40

60

80

100

Fold+expression

3A+CDVehicle'ControlDrug'CompoundControls*

*

0

20

40

60

80

100

Fold+expression

3A+PEG

*

*

EV

TRIF

TRIM21

TRIF+TRIM21 25 5 1

0.20

20

40

60

80

100


Fold5expression

3A5TFA*

46""

"





following!6hr! incubation.!A,!4A,compound! in! cyclodextrin! (CD)! vehicle! control;! B,,4A,compound, in!

polyethylene!glycol!(PEG)!vehicle!control;!and!C,,4A,compound,in!trifluoroacetic!acid!(TFA)!vehicle!



A

B

C

0

20

40

60

80

100

Fold+expression

4A+CDVehicle'ControlDrug'CompoundControls*

*

0

20

40

60

80

100

Fold+expression

4A+PEG

*

*

EV

TRIF

TRIM21

TRIF+TRIM21 25 5 1

0.20

20

40

60

80

100


Fold5expression

4A5TFA

*

*

47""

Table!3.2:!%!Inhibition!of!Luciferase!Assays.!

""

3.3!BCA!Assay!

The"BCA"assay"was"used"to"determine"the"concentration"of"the"protein,"specifically"Trim21,"for"use"in"the"thermal"denaturation"assay."It"was"vital"to"establish"the"concentration"to"calculate"for"the"volumes"required"for"the"next"step"of"the"project."To"ensure"precise"measurements,"it"was"necessary"to"do"the"standards"and"samples"in"triplicates."

48""

Table!3.3:!BCA!Results!of!the!standards.!

"

Table!3.4:!Protein!concentration!of!protein!samples!determined!by!BCA!Assay.!

""

"

Figure!3.8:!Standard!Curve!of!the!BCA!Standards.!

y"="0.0006x"+"0.1183"R²"="0.99487"

0.0"

0.2"

0.4"

0.6"

0.8"

1.0"

1.2"

1.4"

0" 200" 400" 600" 800" 1000" 1200" 1400" 1600" 1800" 2000"

O.D.&@&570nm

&

Concentration&(μg/ml)&

BCA&Standard&Curve&

49##

3.4$Melt$Curves$

The#thermal#shift#assay#is#fully#discussed#in#Section#2.6.#DSF#was#used#as#a#

screening#method#for#Trim21.#Essentially,#the#technique#relies#on#the#use#of#

specific#dyes,#in#this#case#Sypro#Orange,#that#fluoresce#as#they#interact#with#

protein#in#their#denaturation#transition.#For#the#purpose#of#the#project,#DSF#was#

conducted#to#determine#the#specificity#of#the#compounds,#3A#and#4A,#with#

Trim21#protein#and#IRF3#protein.#Typically#a#curved#graph#is#generated.#The#

shifting#of#the#curve#to#the#left#or#right#of#the#control#decides#the#binding#of#the#

ligands.#A#shift#towards#the#left#indicates#the#destabilisation#of#the#protein#

whereas#a#shift#to#the#right#is#a#sign#of#the#ligand#binding#to#the#protein.#

Generally,#a#minimum#2Jdegree#shift#to#the#right#is#considered#significant.#

#

Before#starting#the#assay,#it#was#essential#to#determine#the#optimal#concentration#

for#our#proteins.#Therefore,#following#determination#of#protein#concentration#

from#BCA#assay,#a#dose#response#of#our#proteins#was#conducted,#as#seen#in#

Figure*3.9#for#Trim21#dose#response#and#Figure*3.10#for#IRF3#dose#response.#

For#both#cases,#it#was#clear#that#the#top#concentration#of#0.45μg/ml#and#

0.5μg/ml,#respectively,#were#the#optimal#concentration#of#the#proteins#to#work#

with.#

#

Following#this,#since#the#compounds#were#solubilised#in#DMSO,#it#was#necessary#

that#a#DMSO#control#with#each#of#the#proteins#should#be#carried#out.#When#DMSO#

is#added#to#the#protein,#the#protein#is#destabilised.#This#can#be#seen#in#Figure*

3.11!for#the#Trim21#protein#and#Figure*3.12#for#the#IRF3#protein.#Once#again,#a#

dose#response#curve#was#established#and#we#decided#to#employ#1%#DMSO#

concentration#to#be#tolerable.#

#

Aside#from#a#DMSO#control,#it#was#also#required#to#do#a#control#without#the#

DMSO#and#compounds.#The#thermal#stability#of#the#proteins#was#then#

established#and#the#results#are#laid#out#in#Figure*3.13#for#the#series#A#

compounds#on#Trim21#protein#and#Figure*3.14#for#the#series#A#compounds#on#

IRF3#protein.#The#DMSO#control#is#our#reference#point#in#this#case#and#as#

50""

discussed"briefly"above,"the"shifting"of"the"curve"is"what"needed"to"be"established.""With"the"DMSO"control"in"red"(refer"to"Figure,3.13),"we"can"see"that"both"3A"and"4A"compounds"have"shifted"to"the"right"greater"than"2Xdegrees."This"suggests"that"the"drug"compounds"are"binding"to"the"Trim21"protein"and"thus"might"contribute"to"the"function"of"Trim21"in"this"purpose,"which"is"to"inhibit"IFNXβ"promoter"activity.""In"contrast,"as"seen"in"Figure,3.14,"the"series"A"compounds"have"shifted"negatively,"or"to"the"left"of"the"DMSO"control"in"red."This"tells"us"that"drug"compounds"are"not"binding"to"the"IRF3"protein,"which"is"what"we"would"expect"since"IRF3"protein"is"promoting"IFNXβ"promoter"activity.!

51""

"

"

Figure!3.9:!Determining!protein!concentrations!of!Trim21!for!further!analysis!in!thermal!shift!assay.!

"

"

Figure!3.10:!Determining!protein!concentrations!of!IRF3!for!further!analysis!in!thermal!shift!assay.!

0"

5000"

10000"

15000"

20000"

25000"

30000"

35000"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"

Fluorescence&Intensity&

Temperature&(˚C)&

Protein&Concentrations&of&Trim21&

0.01"TRIM21"0.05"TRIM21"0.10"TRIM21"0.44"TRIM21"

0"

5000"

10000"

15000"

20000"

25000"

30000"

35000"

40000"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"


Temperature&(˚C)&

Protein&Concentrations&of&IRF3&

0.01"IRF3"0.05"IRF3"0.1IRF3"0.5"IRF3"

52""

"

"

Figure!3.11:!Determining!percentage!of!DMSO!used!in!Trim21!protein!in!thermal!shift!assay.!

"

"

Figure!3.12:!Determining!percentage!of!DMSO!used!in!IRF3!protein!in!thermal!shift!assay.!

0"

20"

40"

60"

80"

100"

120"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"


Temperature&(˚C)&

DMSO&Control&on&Trim21&protein&

CONTROL"0.1%"DMSO"1%"DMSO"2%"DMSO"

0"

20"

40"

60"

80"

100"

120"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"


Temperature&(˚C)&

DMSO&control&on&IRF3&protein&

CONTROL"

0.5%"DMSO"

1%"DMSO"

2%"DMSO"

53""

"

"

Figure!3.13:!Analysis!of!series!A!compounds!on!Trim21!protein!using!thermal!denaturation!assay.!!

"

"

Figure!3.14:!Analysis!of!series!A!compounds!on!IRF3!protein!using!thermal!denaturation!assay.!

0"

20"

40"

60"

80"

100"

120"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"


Temperature&(˚C)&

Series&A&Compounds&on&Trim21&protein&

CONTROL"DMSO"2A"3A"4A"

0"

20"

40"

60"

80"

100"

120"

0" 10" 20" 30" 40" 50" 60" 70" 80" 90" 100"


Temperature&(˚C)&

Series&A&Compounds&on&IRF3&protein&

CONTROL"DMSO"2A"3A"4A"

54""

3.5!Bioinformatics!

Initially"started"looking"at"Trim21"as"the"predicted"gene"target"for"the"project"and"looked"for"potential"miRs"of"interest"as"well"as"diseases"found"to"be"associated"with"the"miRs"(refer"to"Table,3.5)."Following"initial"analysis,"the"miRs"of"interest"were"compared"with"the"Nanostring"Screening"Data"to"determine"fold"significance"in"pSS"patients"versus"SLE"patients"and"only"statistically"significant"miRs"were"picked,"based"on"miRs"of"interest"(refer"to"Table,3.6)."Statistically"significant"means"an"expression"ratio"of">2.0"for"upregulated"miRs"and"<0.5"fold"ratio"for"downregulated"miRs,"as"recommended"by"Nanostring."Appendix,A"shows"the"miRs"of"interest"based"on"random"selection"acquired"from"two"different"database"sites,"miRWalk"(http://www.umm.uniXheidelberg.de/apps/zmf/mirwalk/)"and"miRDB"(http://mirdb.org/miRDB/).""Appendix,B"shows"the"top"30"miRs"from"Nanostring"Screen"after"normalisation"where"two"upregulated"and"two"downregulated"miRs"in"PBMCs"of"pSS"patients"versus"controls"were"picked"from"the"list"for"expression"and"validation"analysis"in"this"project."Table,6.1"shows"the"top"30"overexpressed"miRs"in"pSS"patients"versus"healthy"controls;"while"Table,6.2"shows"the"top"30"reduced"miRs"in"pSS"patients"versus"healthy"controls"together"with"some"of"their"predicted"gene"targets,"their"respective"roles"and"disease"association."Online"tools"including"the"National"Centre"fro"Biotechnology"Information’s"(NCBI)"PubMed"(http://www.ncbi.nlm.nih.gov/pubmed)"and"GeneCards"(http://www.genecards.org)"were"utilised"to"search"for"the"above.""Two"miRs"were"selected"from"each"table"for"further"analysis"and"validation"(refer"to"Table,3.7)."If"the"miRs"are"found"to"be"upregulated"then"we"know"that"their"predicted"gene"targets"should"be"downregulated"suggesting"that"in"disease"context,"they"would"be"antiXinflammatory"genes."If"the"miRs"are"shown"to"be"downregulated,"then"it"is"assumed"that"its"predicted"gene"targets"will"be"upregulated."The"table"lists"out"the"diseases"associated"of"the"genes"and"from"the"reviews"published"by"the"authors"referenced,"suggests"whether"there"is"a"direct"correlation,"as"discussed"above."

55""

Table&3.5:&miRs&of&interest&with&Trim21&as&the&predicted&gene&target&along&with&its&associated&diseases.&

&

56##

Table&3.6:&Expression&ratio&of&the&statistically&significant&miRs&of&interest&from&Nanostring&Screening&

between&patients&and&controls.&

##

Table&3.7:&Upregulated&and&Downregulated&miRs&of&interest.&

#

57##

3.6$Primer$Optimisation$

Following#substantial#bioinformatic#analysis#of#miR#screen#data,#miRs#and#its#predicted#gene#targets#of#interest#were#designed#and#optimised,#as#described#in#Section#2.8,#at#three#different#annealing#temperature#before#examining#their#expression#importance#in#pSS#patients#versus#controls.#The#table#shown#below#(Table)3.8)#lists#the#primer#sequences#of#miRs#and#its#gene#targets.#

3.6.1$Primer$Sequences$

Table$3.8:$List$of$primer$sequences$for$miRs$of$interest$and$its$respective$gene$targets.$

#

58##

3.6.2%Gel%Analysis%of%miRs%on%interest%+%predicted%genes%

In#order#to#validate#the#expression#of#the#miRs#of#interest,#primers#were#optimised#and#analysed#on#1%#agarose#gels#following#primer#design.#The#primers#for#each#of#the#miRs#and#genes#were#optimised#for#three#different#annealing#temperatures#starting#at#50˚,#55˚,#and#62˚C.#As#seen#in#Figure'3.15,#miRF30e#produced#distinct#single#bands#at#each#temperature#but#based#on#its#amplicon#size#at#70bp,#55˚C#was#the#closest#annealing#temperature.#For#miRF125a,#two#bands#can#be#seen#for#50˚#and#55˚#that#could#potentially#be#primer#dimers#but#at#62˚,#a#single#thick#band#was#produced#around#~70bp#which#is#at#its#appropriate#position#(71bp).##Figure'3.16#also#shows#single#distinct#bands#for#each#of#the#miR,#194#and#98,#respectively.#For#miRF194,#62˚#was#picked#as#the#annealing#temperature#as#it#was#the#closest#to#its#corresponding#location#at#59bp#and#55˚#for#miRF98,#again#for#its#reciprocal#location#size#at#58bp.#The#respective#genes#for#the#miRs#were#also#analysed#at#three#annealing#temperatures#with#all#genes#with#a#significant#annealing#temperature#at#62˚C#(refer#to#Figure'3.17).##

#

Figure% 3.15:% Optimisation% of% miR@30e% and% miR@125a% at% 50˚,% 55˚,% and% 62˚C% anneling% temperatures%

using% sterile% nuclease@free% water% and% control% cDNA% and% 1kb% DNA% ladder% visualised% by% SyproRed%

staining%on%a%1%%agarose%gel.%%

59##

#

#

Figure%3.16:%Optimisation%of%miR@194%and%miR@98%at%50˚,%55˚,%and%62˚C%annealing%temperatures%using%

sterile%nuclease@free%water%and%control%cDNA%and%1kb%DNA%ladder%visualised%by%SyproRed%staining%

on%a%1%%agarose%gel.%

#

Figure%3.17:%Optimisation%of% the%predicted%gene% targets%of%miRs%of% interest% including%CXCL3%at%62˚,%

65˚,%and%68˚C%annealing%temperature%and%JAK3,%IL10,%and%SOCS1%at%62˚C%annealing%temperature%using%

sterile%nuclease@free%water%and%control%cDNA%and%1kb%DNA%ladder%visualised%by%SyproRed%staining%

on%a%1%%agarose%gel.%

60##

3.7$mRNA$concentration$in$pSS$Patients$and$Healthy$Controls$

PBMCs#of#patient#and#control#samples#were#isolated#by#density#gradient#

centrifugation.#mRNA#of#the#samples#were#isolated#using#TRIzol#reagent#and#

quantified#as#discussed#in#Section#2.11.#The#concentration#of#patient#and#control#

samples#shown#in#Table)3H)are#average#of#two#readings#and#the#microgram#

amount#of#mRNA#required#for#cDNA#conversion#was#calculated#using#these#

values#for#realGtime#quantitative#analysis.#

Table$3.9:$mRNA$concentration$in$pSS$patients$and$healthy$controls$using$NanoDrop$

Spectrophotometer.$

Patient) Concentration)(ng/μl))RVPT3B# 479.45#RVPT8# 413.5#RVPT9# 467.95#RVPT10# 421.55#RVPT11# 443.05#RVPT13# 430.75#RVPT14# 542#RVPT20# 203.1#RVPT21# 211.75#

Control) Concentration)(ng/μl))RVCTL3B# 454.05#RVCTL8# 518.15#RVCTL9# 554.8#RVCTL10# 420.7#RVCTL11# 442.6#RVCTL13# 462.7#RVCTL20# 423.75#RVCTL21# 265.5#

#

61##

3.8%Expression%of%miRs%of%interest%in%pSS%Patients%vs%Healthy%Controls%

Following#primer#design#and#optimisation#for#miRs#of#interest#and#determining#

the#respective#annealing#temperature,#a#realFtime#quantitative#analysis#was#

performed#for#the#patient#and#control#samples#provided,#as#detailed#in#Section#

2.13.#Statistical#analysis#and#determination#of#significance#were#calculated#using#

GraphPad#Prism#6#(version#6.0f).#Prior#to#determining#statistical#significance,#all#

data#were#normalised#on#Microsoft#Excel.#

#

RealFtime#analysis#shown#in#Figure'3.18#presents#the#expression#of#miRF30e#in#

PBMCs#of#pSS#patients#vs#healthy#controls.#Although#it#has#not#shown#to#be#

statistically#significant#(p<0.05),#the#graph#shows#that#miRF30e#is#slight#

upregulated#and#comparing#it#to#the#screening#analysis,#this#confirms#that#miRF

30e#is#considered#to#be#upregulated#in#pSS#patients.#

#

The#next#three#graphs#posted#below#are#all#downregulated#miRs.#Since#the#

screening#results#were#off#pooled#samples,#it#was#necessary#to#validate#each#miR#

in#individual#samples#from#pSS#patients#and#healthy#controls#to#ensure#

representative#trend.#Figure'3.19#displays#miRF125a#expression,#however,#based#

on#these#findings,#it#seems#that#miRF125a#is#upregulated#rather#than#

downregulated#as#proposed#from#the#screening#results.#The#expression#of#miRF

194#can#be#seen#in#Figure'3.20#and#displays#a#decreased#expression#in#pSS#

patients,#as#hoped#based#on#initial#analysis.#However,#it#was#not#deemed#to#be#

statistically#significant#(p<0.05).#

#

Figure'3.21#shows#miRF98#expression#believed#to#be#downregulated#in#pSS#

patients#compared#to#healthy#controls.#RealFtime#analysis#presents#a#slight#

decreased#in#expression#in#pSS#patients#but#not#considered#statistically#

significant#(p<0.05).#RNU6B#was#used#as#the#reference#miR#endogenous#control#

in#qPCR#analysis#of#our#miRs.#

62##

#

Figure%3.18:%Expression%levels%of%miR@30e%in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%miR@

30e% raw% spectrum%expression%with% a%pBvalue% of% 0.5714.% B,%miR@30e% expression%minus% the% outliers%

with%a%pBvalue'of%0.6571.%Endogenous%RNU6B%was%used%as%reference%control%for%miR%expression.%

#

#

Figure%3.19:%Expression%levels%of%miR@125a%in%PBMCs%of%pSS%patient%versus%healthy%controls.%%A,%miR@

125a%raw%spectrum%expression%with%a%pBvalue'of%0.3754.%B,%miR@125a%expression%minus%the%outliers%

with%a%pBvalue%of%0.6667.%Endogenous%RNU6B%was%used%as%reference%control%for%miR%expression.%

#

pSS control-5

0

5

10

15

20

Fo

ld e

xp

re

ss

ion

miR-30e

p = 0.5714

pSS control0.0

0.5

1.0

1.5

Fo

ld e

xp

re

ss

ion

miR-30e

p = 0.6571A B

pSS control-5

0

5

10

15

Fo

ld e

xp

re

ss

ion

miR-125a

p = 0.3754

pSS control0.0

0.5

1.0

1.5

2.0

Fo

ld e

xp

re

ss

ion

miR-125a

p = 0.6667

A B

63##

#

Figure%3.20:%Expression%levels%of%miR@194%in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%miR@

194%raw%spectrum%expression%with%a%pBvalue%of%0.7023.%B,%miR@125a%expression%minus%the%outliers%

with%a%pBvalue%of%>0.9999.%Endogenous%RNU6B%was%used%as%reference%control%for%miR%expression.%

#

#

Figure%3.21:%Expression%levels%of%miR@98%in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%miR@98%

raw%spectrum%expression%with%a%pBvalue%of%0.8983.%B,%miR@98%expression%minus%the%outliers%with%a%pB

value%of%0.5238.%Endogenou%RNU6B%was%used%as%reference%control%for%miR%expression.%

pSS control-2

0

2

4

6

8

10

Fo

ld e

xp

re

ss

ion

miR-194

p = 0.7023

pSS control0.0

0.5

1.0

1.5

2.0

Fo

ld e

xp

re

ss

ion

miR-194

p = > 0.9999A B

pSS control0.0

0.5

1.0

1.5

2.0

Fo

ld e

xp

re

ss

ion

miR-98

p = 0.8983

pSS control0.6

0.8

1.0

1.2

Fo

ld e

xp

re

ss

ion

miR-98

p = 0.5238

A B

64##

3.9%Expression%of%Predicted%Gene%Targets%of%miRs%of%Interest%

Subsequent#to#a#realFtime#analysis#of#the#miRs,#the#expression#of#the#predicted#targets#of#these#miRs#was#achieved#for#the#patient#and#control#samples#provided,#as#detailed#in#Section#2.13.#Once#again,#statistical#analysis#and#determination#of#significance#were#calculated#using#GraphPad#Prism#6#(version#6.0f).#Prior#to#determining#statistical#significance,#all#data#were#normalised#on#Microsoft#Excel.##SOCS1#gene#as#seen#in#Figure'3.22,#is#a#predicted#target#of#miRF30e.#Since#miRF30e#is#regarded#as#an#upregualted#miR,#we#would#expect#expression#of#SOCS1#gene#in#pSS#patients#vs#healthy#controls#to#be#decreased#and#from#the#findings,#this#seems#to#be#the#case,#but#not#considered#to#significant#(p<0.05).#CXCL3#gene#and#JAK3#gene#are#both#predicted#targets#of#miRF194.#Their#expressions#are#respectively#shown#in#Figure'3.23#and#Figure'3.24.#Since#miRF194#is#a#downregulated#miR,#we#would#expect#that#both#genes#to#be#increased#in#pSS#patients#expression#compared#to#healthy#controls.#While#CXCL3#gene#expression#is#shown#to#be#increase#in#pSS#patients,#however#in#this#case,#JAK3#gene#expression#is#considerably#reduced#in#pSS#patients#although#not#significant#(p<0.05).###ILF10#gene#expression#is#displayed#in#Figure'3.25;#a#predicted#target#of#of#miRF98#is#increased#in#pSS#patients.#miRF98#is#also#a#downregulated#miR#from#bioinformatic#analysis,#which#would#suggest#that#ILF10#gene#expression#should#be#increase#in#pSS#patients,#which#is#the#case#here.#18s#was#used#as#the#reference#gene#endogenous#control#in#qPCR#analysis#of#our#target#genes.##Since#we#didn’t#have#enough#cDNA#samples#left,#we#could#only#analyse#expression#of#JAK3#gene#and#IL10#gene#on#three#patient#samples#and#three#control#samples.#It#is#important#to#note#that#in#order#to#accurately#validate#expression#in#patients#vs#controls,#a#number#of#samples#are#generally#required#for#analysis.#

65##

#

Figure%3.22:%Expression% levels%of% SOCS1% in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,% SOCS1%

gene%raw%spectrum%expression%with%a%pBvalue%of%0.9319.%B,%SOCS1%gene%expression%minus%the%outliers%

with%a%pBvalue%of%0.6277.%Endogenous%18s%was%used%as%reference%control%for%gene%expression.%

#

#

Figure%3.23:%Expression% levels%of%CXCL3% in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%CXCL3%

gene%raw%spectrum%expression%with%a%pBvalue%of%0.5604.%B,%CXCL3%gene%expression%minus%the%outliers%

with%a%pBvalue%of%0.7706.%Endogenous%18s%was%used%as%reference%control%for%gene%expression.%

##

pSS control-500

0

500

1000

Fo

ld e

xp

res

sio

nSOCS1 gene

p = 0.9319

pSS control-0.5

0.0

0.5

1.0

1.5

Fo

ld e

xp

res

sio

n

SOCS1 genep = 0.6277

A B

pSS control-100

0

100

200

300

Fo

ld e

xp

res

sio

n

CXCL3 gene

p = 0.5604

pSS control-2

0

2

4

6

Fo

ld e

xp

res

sio

n

CXCL3 gene

p = 0.7706A B

66##

#

Figure%3.24:%Expression%levels%of%JAK3%in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%JAK3%gene%

raw%spectrum%expression%with%a%pBvalue%of%0.4000.%B,%JAK3%gene%expression%minus%the%outliers%with%a%

pBvalue%of%0.3333.%Endogenous%18s%was%used%as%reference%control%for%gene%expression.%

#

#

Figure%3.25:%Expression%levels%of%IL10%in%PBMCs%of%pSS%patient%versus%healthy%controls.%A,%IL10%gene%

raw%spectrum%expression%with%a%pBvalue%of%0.4000.%Endogenous%18s%was%used%as%reference%control%

for%gene%expression.%

##

pSS control0.00

0.02

0.04

0.06

0.08

0.10

Fo

ld e

xp

re

ss

ion

JAK3 gene

p = 0.4000

pSS control0.00

0.02

0.04

0.06

0.08

0.10

Fo

ld e

xp

re

ss

ion

JAK3 gene

p = 0.3333

A B

pSS control-1

0

1

2

3

4

Fold

exp

ress

ion

IL10 gene

p = 0.4000

A

67##

4.0%Discussion%

4.1%Luciferase%Assays%

Trim21,#an#interferonFinducible#protein,#is#frequently#targeted#by#autoantibodies#not#just#in#patients#with#primary#SS#but#also#in#other#autoimmune#disorders#including#SLE.#The#TRIM#family#of#proteins#have#been#branded#as#key#negative#regulators#(Narayan#et#al.,#2014)#of#responses#mediated#by#interferon#regulatory#factors#(IRF).#A#role#for#TRIMs#as#regulators#of#transcription#factor#activity,#localisation#and#stability#is#emerging.#Evidence#suggests#that#Trim21#targets#and#regulates#the#stability#of#several#members#of#the#IRF#family#(Jefferies#et#al.,#2011).#Trim21#has#been#known#to#mediate#the#ubiquitination#of#several#members#of#the#IRF#transcription#factor#family,#essential#transcription#factors#in#the#induction#of#cytokine#expression#(Espinosa#et#al.,#2011).#Type#I#interferons#(IFNFα#and#Fβ)#are#produced#following#a#bacterial#or#viral#infection#which#is#central#to#innate#immune#responses.#Specific#families#of#IRF#transcription#factors,#IRF3#and#IRF7,#have#been#shown#to#be#essential#for#type#I#IFN#gene#induction#(Higgs#et#al.,#2008)#in#response#to#the#recognition#of#pathogens#through#transmemebrane#tollFlike#receptors#(TLRs)#or#cytosolic#pattern#recognition#receptors#(PRRs).#As#discussed#above,#Trim21#has#been#identified#as#an#E3#ubiquitin#ligase#and#that#it#is#capable#of#switching#off#and#limiting#the#production#of#type#I#IFN#following#viral#or#bacterial#infection.##Previous#experimental#studies#in#the#lab#have#focused#on#the#regulation#of#Trim21#to#IFNFβ#production#(Higgs#et#al.,#2008;#Espinosa#et#al.,#2009).#They#have#shown#that#IRF3#and#IRF7#interact#with#Trim21#following#activation#of#TLR3#and#TLR7/9,#respectively.#This#interaction#ultimately#leads#to#termination#of#transcription#of#IRFFregulated#type#I#IFNs#and#proinflammatory#cytokines.#IRF3#is#a#constitutively#expressed#member#of#the#IRF#family#that#regulates#the#primary#induction#of#IFNFβ#in#response#to#viral#and#bacterial#infection#downstream#of#TLR3,#TLR4#and#cytosolic#PRRs#(Mogensen,#2009).#Both#TLR3#and#TLR4#induce#type#I#IFN#production#in#a#similar#fashion,#through#recruitment#and#activation#of#the#TIR#domain#containing#adaptor#inducing#IFN$β#(TRIF).#Defects#in#the#

68##

expression#or#activity#of#Trim21#may#contribute#to#the#pathogenic#

overproduction#of#IFNs#observed#in#SLE#since#Trim21#has#been#suggested#to#

have#an#important#role#in#regulating#both#proFinflammatory#and#type#I#IFN#

responses#downstream#of#TLRs#(Jefferies#et#al.,#2011).#

#

As#discussed,#Trim21#has#been#found#to#be#a#negative#regulator#of#innate#

immune#signalling#through#the#interaction,#ubiquitination,#and#subsequent#

proteasomal#degradation#of#the#transcription#factors#IRF3#and#IRF7#(Oke#and#

WahrenFHerlenius,#2012).#By#ubiquitinating#and#targeting#IRF3#and#IRF7#for#

degradation,#Trim21#effectively#turns#off#the#production#of#type#I#IFNs,#thus#

preventing#overproduction#of#this#potentially#pathogenic#cytokine.#

#

The#present#therapeutics#of#SLE#focus#very#much#on#the#treatment#of#symptoms#

of#the#disease#rather#than#the#underlying#causative#effects,#namely#the#

overproduction#of#type#I#IFNs#due#to#the#recognition#of#self#RNA#and#DNA.#

Previous#studies#carried#out#by#the#lab#group#have#demonstrated#a#role#for#

Trim21#in#negatively#regulating#type#I#IFN#levels.#The#project#looked#at#the#effect,#

if#any,#of#drug#compounds#on#the#ability#of#Trim21#to#negatively#regulate#IFNFβ#

promoter#activity.#The#adaptor#protein,#TRIF#was#used#to#drive#the#promoter#in#

these#experiments,#as#IRF3#and#IRF7#lie#downstream#of#this#pathway.#These#drug#

compounds#were#used#in#a#luciferase#reporter#assay#to#determine#the#affect#that#

these#compounds#have#on#the#activity#of#Trim21.#The#objective#was#to#determine#

the#effect#of#these#compounds#on#the#activity#of#Trim21#as#in#inhibitor#of#IFNFβ#

production.#

#

HEK293FT#cells#were#used#in#this#project#due#to#their#nature#of#growing#very#

easily#as#well#as#readily#transfection#that#has#been#widely#used#in#research#and#

cellular#biology.#The#HeLa#cell#line#has#previously#been#used#in#the#lab#that#

resulted#in#no#inhibition#of#IFNFB#promoter#activity.#Accordingly,#HEK293FT#cells#

were#seeded#at#1x105#cells/ml#and#transfected#with#plasmids#containing#TRIF#

(100ng),#P125#(100ng),#Renilla#luciferase#(100ng),#and#EV#(100ng),#together#

with#Trim21#(100ng).#18#hours#postFtransfection#the#cells#were#treated#with#

increasing#concentrations#of#relevant#compounds#for#6#hours.#Following#

69##

treatment#of#the#cells#with#the#relevant#small#compounds,#cells#were#lysed#and#the#luciferase#reporter#assay#was#carried#out#in#order#to#determine#the#amount#of#IFNFβ#promoter#activity#in#the#cell.##

These#small#molecule#compounds#have#been#designed#to#potentially#increase#the#activity#of#Trim21#in#order#to#further#inhibit#the#production#of#downstream#type#I#IFNs.#In#testing#the#compounds#using#the#luciferase#reporter#assay,#the#inhibition#of#IFNFβ#promoter#activity#inversely#correlates#to#the#level#of#Trim21#activity.#Therefore,#a#reduction#in#IFNFβ#promoter#activity#corresponds#to#an#increase#in#Trim21#activity.#Hypothetically,#we#would#expect#to#observe#an#increased#inhibition#of#IFNFβ#promoter#activity#upon#the#treatment#of#transfected#cells#with#the#drug#compounds,#corresponding#to#increase#Trim21#activity.#However,#we#observed#variability#in#the#results#with#the#drug#compounds#even#though#there#was#inhibition#of#IFNFβ#promoter#activity.#It#was#also#observed#that#over#all#the#drug#compounds,#there#was#no#dose#dependent#response#in#activity#of#Trim21.#It#was#expected#that#with#an#increase#in#compound#dosage,#an#increase#in#Trim21#activity#would#also#be#observed.#The#differences#in#the#cell#response#to#the#treatment#with#different#compounds#are#likely#attributable#to#a#number#of#things#including#mycoplasma#infection,#human#errors#such#as#pipetting#and#calculations,#and#the#concentration#nature#of#the#diluents,#especially#NaOH,#where#a#higher#percentage#of#NaOH#can#alter#the#pH#of#the#cell,#which#can#lead#to#cell#death.##

As#this#is#still#an#ongoing#project,#the#group#have#planned#to#modify#the#structure#of#the#compounds#and#to#employ#the#modified#compounds#through#luciferase#reporter#assay.#There#is#also#the#ongoing#problem#of#the#HEK293FT#cell#line#where#we#have#noticed#a#greater#susceptibility#to#infection.#We#tried#to#minimise#possible#infection#by#ensuring#the#fume#hood#and#incubator#was#cleaned#each#week,#the#use#of#own#medium#and#reagents,#and#filtering#the#medium#onto#sterile#falcon#tubes.#It#was#suspected#that#the#HEK293FT#cell#line#was#infected#with#mycoplasma.#Although#mycoplasma#does#not#typically#harm#cell#cultures,#they#exist#typically#at#high#concentrations#in#cell#cultures#thereby#competing#for#essential#nutrients#with#the#host#cells,#which#results#in#altered#growth.#

70##

Mycoplasma#infections#are#known#to#induce#a#myriad#of#cellular#changes#leading#

to#false#interpretation#of#experimental#results#and#significant#research#data.#

Experimental#variability#causes#differences#in#cell#viability#or#transfection#

efficiency.#Another#typical#source#of#variability#in#data#is#human#error#especially#

pipetting.#Problems#persisted#with#the#miniscule#volumes#used.#

#

By#targeting#an#increase#in#activity#of#Trim21,#theoretically#this#will#inhibit#IFNFβ#

promoter#activity,#subsequently#reducing#the#inflammatory#response#(Stacey#et#

al.,#2012)#from#self#RNA#and#DNA.#The#taking#of#a#small#compound#based#

therapeutic#rather#than#a#number#of#assorted#nonFsteroidal#antiFinflammatory#

drugs#(NSAIDS)#to#combat#this#inflammatory#response#(Petschow#et#al.,#2015)#

would#also#increase#a#patient’s#quality#of#life.#

#

The#overall#hypothesis#of#the#lab#is#that#by#increasing#the#activity#of#Trim21#that#

it#is#also#increasing#its#ability#to#reduce#type#I#IFN#production#thus#reducing#one#

of#the#main#symptoms#and#causes#of#the#disease.#The#group#hopes#that#a#

patentable#product#may#be#developed#in#the#near#future#as#novel#therapeutic#

treatment#for#SLE.#

#

4.2%Thermal%Shift%Assay%

The#thermal#shift#was#conducted#to#see#the#effect#the#drug#compounds,#3A#and#

4A,#have#on#the#thermal#stability#of#the#proteins,#Trim21#and#IRF3.#Typically#a#

curved#graph#is#generated#associated#with#a#shift,#both#positive#and#negative.#The#

shifting#of#the#curve#to#the#left#or#right#of#the#reference#control#decides#the#

binding#of#the#ligands.#A#shift#towards#the#left#indicates#the#destabilisation#of#the#

protein#whereas#a#shift#to#the#right#is#a#sign#of#the#ligand#binding#to#the#protein.#

Generally,#a#minimum#2Fdegree#shift#to#the#right#is#considered#significant.#A#

positive#shift#of#the#curve#tells#us#that#the#proteins#have#increased#stability.#

#

The#thermal#shift#assay#is#an#inexpensive,#simple#and#quick#method#(Vollrath#et#

al.,#2014)#that#can#be#performed#on#a#readily#available#realFtime#PCR#machine.#It#

71##

combines#sample#temperature#control#and#dye#fluorescence#detection#(Kranz#and#SchalkFHihi,#2011)#to#achieve#a#graphed#curve.##In#our#case,#since#Trim21#has#been#suggested#to#inhibit#IFNFβ#promoter#activity,#the#binding#of#the#drug#compounds#to#the#Trim21#protein#in#the#form#of#a#shift#in#the#curve#could#suggest#that#these#compounds#might#be#increasing#the#inhibition#of#IFNFβ#promoter#activity.#On#the#other#hand,#the#IRF3#protein#is#known#to#promote#IFNFβ#promoter#activity#suggesting#that#the#drug#compounds#should#not#bind#to#this#protein.##Based#on#the#results#presented#in#Section#3.4,#we#achieved#the#expected#results#for#both#3A#and#4A#compounds.#Both#compounds#have#been#found#to#have#a#positive#shift#following#binding#to#Trim21#protein#indicating#a#potential#increase#in#Trim21#function#to#inhibit#IFNFβ#promoter#activity.#In#comparison,#both#compounds#had#a#negative#shift#when#bound#to#IRF3#protein.##

4.3%Bioinformatics%

The#project#initially#began#with#bioinformatic#analysis#of#Trim21#as#our#predicted#gene#target.#Following#this,#the#predicted#miRs#that#target#Trim21#were#assessed#and#ten#random#miRs#were#chosen#for#initial#analysis.#These#chosen#miRs#were#compared#with#the#screening#results#conducted#by#Nanostring#to#look#for#expression#in#pSS#patients#versus#SLE#patients#and#results#were#recorded.##From#the#same#screening#results,#we#normalised#the#top#30#miRs#that#have#been#shown#to#be#increased#and#decreased#in#pSS#patients,#as#seen#in#Appendix'B.#From#our#top#30#lists#of#miRs,#we#further#analysed#two#miRs#from#each#respective#table#for#realFtime#investigation#of#individual#samples#of#pSS#patients#vs#healthy#controls#that#have#previously#shown#differential#expression.##Expression#of#the#miRs#of#interest#as#well#as#their#corresponding#gene#targets#are#shown#and#discussed#in#Section#3.8#and#3.9,#respectively.#As#discussed,#

72##

downregulated#miRs#in#pSS#patients#versus#healthy#controls#suggest#that#its#predicted#gene#targets#should#be#upregulated,#whereas#if#a#miR#is#shown#to#be#upregulated,#its#gene#targets#should#be#downregulated.##Because#of#the#limited#samples#of#patient#and#control#cDNA,#it#was#not#at#a#luxury#to#test#the#expression#of#each#of#the#predicted#targets#of#the#miRs.##miR$98'

The#expression#of#miRC98#was#expected#to#be#downregulated#in#pSS#patients#compared#to#healthy#controls.#RealCtime#validation#demonstrated#a#similar#trend#in#underexpression#of#miRC98#in#pSS#patients#vs#healthy#controls.#Interleukin#(ILC10),#which#is#an#immunoregulatory#cytokine,#plays#a#vital#role#in#inflammatory#and#immune#reactions.#ILC10#is#known#to#inhibit#the#synthesis#of#a#number#of#cytokines#such#as#IFN#γ,#IL2,#IL3#and#tumour#necrosis#factor#(TNF)#produced#by#activated#macrophages#and#by#helper#TCcells#(Jabs#et#al.,#2001).#ILC10#have#been#found#to#be#of#high#levels#in#autoimmune#diseases#(Beebe#et#al.,#2002)#such#as#SLE#or#Multiple#Sclerosis#(MS)#as#well#as#in#various#tumours#including#melanoma#(Huang#et#al.,#1999)#and#breast.#ILC10#has#been#demonstrated#to#be#frequently#present#in#renal#cell#carcinoma#tumours#(Wang#et#al.,#1995).#Based#on#these#findings,#we#expect#ILC10#gene#would#be#overexpressed#in#pSS#patients#vs#controls,#which#is#the#case#but#it#was#not#deemed#significant.#In#addition#to#ILC10,#IGF1#is#known#to#be#a#direct#target#of#miRC98.#IGF1#have#been#associated#with#growth#hormone#deficiencies#with#persistent#elevation#(Lim#et#al.,#2007)#of#IGF1.##miR$194'

miRC194#was#shown#to#be#downregulated#with#a#ratio#of#0.42#for#patients#vs#controls#and#was#chosen#as#one#of#the#miRs#of#interest#due#to#its#predicted#association#with#proinflammatory#genes.#Analysis#of#realCtime#investigation#resulted#in#decreased#expression#in#pSS#patients#compared#to#healthy#controls.#Proinflammatory#genes#includes#IL17D,#which#have#been#shown#to#stimulate#interleukin#production#specifically#IL6#and#IL8#and#colony#stimulating#factor#CSF2#in#endothelial#cells#(Brocker#et#al.,#2010).#This#family#of#interleukins#have#been#linked#to#several#autoimmune#diseases#including#RA#and#SLE.#CXCL3#is#a#

73##

chemokine#ligand#and#also#a#predicted#target#of#miRC194#that#regulates#

monocyte#migration#and#adhesion.#It#is#found#as#an#oncogene#in#colon#cancer#(Li#

et#al.,#2004)#and#have#been#found#to#be#express#significantly.#From#our#test,#

CXCL3#was#found#to#be#increase#in#expression#as#what#we#would#expect.#Finally#

JAK3,#which#is#expressed#in#natural#killer#cells,#T#cells#and#B#cells,#is#another#

predicted#gene#target#of#miRC194#that#functions#in#signal#transduction.#Mutations#

in#this#gene#have#been#positively#found#in#severe#combined#immunodeficiency#

and#inflammatory#bowel#disease#sufferers#(Yao#et#al.,#2006).#However,#following#

realCtime#analysis,#JAK3#was#found#to#have#increased#expression#in#pSS#patients#

versus#healthy#controls#rather#than#the#expected#underexpression.#

#

miR$125a'

Reduced#expression#of#miRC125a#in#pSS#has#been#associated#with#CFTR#and#E2F3#

as#its#predicted#targets.#RealCtime#investigation#did#not#agree#with#initial#

bioinformatics#validation#since#miRC125a#expression#was#overexpressed#in#pSS#

patients.#Mutations#in#the#CFTR#gene#have#been#positively#associated#with#cystic#

fibrosis#(Groman#et#al.,#2002).#E2F3#is#a#proteinCencoding#gene#that#regulates#the#

expression#of#genes#involved#in#the#cell#cycle.#Diseases#associated#with#E2F3#are#

mostly#cancers#with#significant#expression#in#bladder#(Olsson#et#al.,#2007),#

prostate#(Foster#et#al.,#2004),#and#retinoblastoma#(Orlic#et#al.,#2006).#

Unfortunately,#no#primers#for#miRC125a#gene#targets#were#ordered#on#time#and#

thus,#no#expression#were#considered#in#pSS#patients#versus#healthy#controls.#

#

miR$30e'

The#predicted#targets#of#miRC30e#include#immuneCrelated#genes#such#as#CD80#

and#SOCS1.#miRC30e#from#initial#bioinformatics#analysis#revealed#to#be#

upregulated#and#was#confirmed#to#be#overexpressed#by#realCtime#validation#to#

be#the#case.#CD80#is#involved#in#the#costimulatory#signal#essential#for#the#

activation#of#TClymphocytes.#Some#of#the#associated#diseases#of#CD80#have#been#

reportedly#found#in#decreased#numbers#in#systemic#autoimmune#diseases#(Wong#

et#al.,#2005)#including#SLE#and#RA.#As#well#as#autoimmune#diseases,#CD80#have#

been#shown#to#be#express#in#low#levels#in#melanoma#patients#(Bernsen#et#al.,#

2003).#This#suggests#that#CD80’s#function#in#the#control#of#melanoma#is#

74##

somewhat#decreased#or#reduced.#SOCS1#is#another#predicted#target#of#miRC30e#that#is#involved#in#the#regulation#of#cytokine#signal#transduction.#In#this#case,#SOCS1#gene#expression#in#pSS#patients#versus#healthy#controls#showed#decreased#expression,#as#hoped,#but#not#considered#statistically#significant.#It#is#commonly#associated#with#various#tumours#of#reduced#expression#in#colorectal#cancer,#hepatocellular#carcinoma#(Okochi#et#al.,#2003)#and#in#chronic#myeloid#leukaemia.##miR$574'

miRC574#is#another#upregulated#miR#from#the#list#but#have#not#been#validated#further#due#to#no#primers#were#ordered#on#time.#It#is#associated#positively#with#MACC1,#which#is#a#key#regulator#of#hepatocyte#growth#factor#HGFCMET#signalling.#Reports#suggest#that#expression#of#MACC1#in#colon#cancer#is#negatively#regulated#by#miRC574#to#suppress#CRC#liver#metastasis#(Cui#et#al.,#2014).##Extensive#bioinformatic#analysis#has#revealed#the#dysregulated#expression#of#certain#miRs#and#their#predicted#gene#targets#in#pSS#patients#versus#healthy#controls.#Since#non#of#the#miRs#nor#the#gene#targets#were#deemed#significant,#they#require#further#investigation#to#confirm#their#legitimacy#in#expression#in#pSS#patients.#Nonetheless,#studies#into#miR#dysregulation#in#disease#processes#should#aid#in#the#development#of#novel#miR#therapeutics.

75##

5.0%Bibliography%#

Alevizos,#I.,#Alexander,#S.,#Turner,#R.#J.#&#Illei,#G.#G.#MicroRNA#expression#profiles#

as#biomarkers#of#minor#salivary#gland#inflammation#and#dysfunction#in#

Sjögren's#syndrome.#Arthritis#Rheum#2011;#63:#535F44.##

Alevizos,#I.#&#Illei,#G.#G.#MicroRNAs#in#Sjögren's#syndrome#as#a#prototypic#

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Alvarez,#M.#L.,#Khosroheidari,#M.,#Eddy,#E.#&#Kiefer,#J.#Role#of#microRNA#1207F5P#

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extracellular#matrix#accumulation#in#the#kidney:#implications#for#diabetic#

nephropathy.#PLoS#One#2013;#8:#e77468.##

Amarilyo,#G.#&#La#Cava,#A.#miRNA#in#systemic#lupus#erythematosus.#Clin#Immunol#2012;#144:#26F31.#

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Bayetto,#K.#&#Logan,#R.#M.#Sjögren's#syndrome:#a#review#of#aetiology,#

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Beebe,#A.#M.,#Cua,#D.#J.#&#de#Waal#Malefyt,#R.#The#role#of#interleukinF10#in#

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sclerosis#(MS).#Cytokine#Growth#Factor#Rev#2002;#13:#403F12.##

Bernsen,#M.#R.,#Hakansson,#L.,#Gustafsson,#B.,#Krysander,#L.,#Rettrup,#B.,#Ruiter,#D.#

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Cain,#H.#C.,#Noble,#P.#W.#&#Matthay,#R.#A.#Pulmonary#manifestations#of#Sjögren's#

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6.0%Appendices%

Appendix%A%

#

Figure%6.1:%Gene%6737%(Trim21)%wass%predicted%to%be%targeted%by%21%miRNAs%in%miRDB.%

#

Figure% 6.2:% Predicted%miRNA% sites% of% Trim21% on%mRNA% 3'% UTR% region% produced% by%miRWalk% and%

other%programs.%

Overexpressed miRs pSS Fold SLE Fold pSS PBMCs pSS CECs Predicted Gene Targets Gene Description Roles Disease Association

hsa-‐miR-‐1283 232.62 0.41 -‐ -‐ CDH2 Cadherin-‐2 Imparting to cells the ability to adhere in a homophilic manner Preeclampsia

BCL2L11 Bcl-‐2-‐like protein 11 Induces apoptosis and anoikis

RAD50 RAD50 Homolog (S. Cerevisiae)Plays a central role in double-‐strand break (DSB) repair, DNA recombination, maintenance of

telomere integrity and meiosishsa-‐miR-‐636 46.52 0.56 0.96 0.97 SNF8 ESCRT-‐II Complex Subunit Play a role in transcription regulation Dilated cardiomyopathy

GTSF1 Gametocyte Specific Factor 1 -‐ Hepatocellular carcinoma

HAPLN1 Hyaluronan And Proteoglycan Link Protein 1 Stabilises the aggregates of proteoglycan monomers

hsa-‐miR-‐1253 44.58 0.41 -‐ -‐ ATP9A ATPase, Class II, Type 9A -‐ Gastrointestinal cancerWDR68 WD repeat-‐containing protein 68 Involved in craniofacial developmentARIH2 Ariadne RBR E3 Ubiquitin Protein Ligase 2 Probable E3 ubiquitin-‐protein ligase

hsa-‐miR-‐143-‐3p 11.15 12.63 -‐ -‐ -‐ -‐ -‐ NephroblastomaChronic fatigue syndrome

hsa-‐miR-‐320e 6.06 0.8 0.74 1.05 -‐ -‐ -‐ Hypertrophic cardiomyopathy

hsa-‐miR-‐369-‐5p 6.01 0.49 -‐ -‐ DHX15 DEAH (Asp-‐Glu-‐Ala-‐His) Box Helicase 15Pre-‐mRNA processing factor involved in

disassembly of spliceosomes after the release of mature mRNA

Pancreatic ductal adenocarcinoma

MACF1 Microtubule-‐Actin Crosslinking Factor 1Plays a role in cross-‐linking actin to other cytoskeletal proteins and also binds to

microtubulesBronchiolitis obliterans syndrome

FRK Fyn-‐Related Kinase Negatively regulates cell proliferation

hsa-‐miR-‐513a-‐3p 5.67 0.43 -‐ -‐ KCNE3 Potassium Voltage-‐Gated Channel, Isk-‐Related Family, Member 3

Regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction,

and cell volume

Hypertrophic cardiomyopathy

RANBP9 RAN Binding Protein 9May act as an adapter protein to couple

membrane receptors to intracellular signaling pathways

Non-‐small cell lung cancer

ARL4C ADP-‐Ribosylation Factor-‐Like 4C May play a role in cholesterol transport

hsa-‐miR-‐597 5.07 0.62 -‐ -‐ TTC8 Tetratricopeptide Repeat Domain 8 Function as a coat complex required for sorting of specific membrane proteins to the primary cilia Immune thrombocytopenic purpura

MYST4 MYST histone acetyltransferase (monocytic leukemia) 4)

Involved in both positive and negative regulation of transcription

AMELX Amelogenin, X-‐linked Plays a role in biomineralisationhsa-‐miR-‐124-‐3p 4.58 0.53 -‐ -‐ -‐ -‐ -‐ Polycystic ovary syndrome

Chron's diseasehsa-‐miR-‐3144-‐5p 4.51 0.5 -‐ -‐ -‐ -‐ -‐ Hepatocellular carcinomahsa-‐miR-‐378b 4.31 0.46 -‐ -‐ -‐ -‐ -‐ -‐

hsa-‐miR-‐574-‐5p 4.29 0.7 1.22 0.89 PTPRU Protein Tyrosine Phosphatase, Receptor Type, UMay function in cell proliferation and migration and play a role in the maintenance of epithelial

integrityColorectal cancer

ACTR3 ARP3 Actin-‐Related Protein 3 Homolog (Yeast) Regulates actin polymerisation Non-‐small cell lung cancer

TRIB1 Tribbles Pseudokinase 1 Interacts with MAPK kinases and regulates activation of MAP kinases

hsa-‐miR-‐147a 4.2 0.52 -‐ -‐ -‐ -‐ -‐ Diffuse large B-‐cell lymphoma

hsa-‐miR-‐125a-‐3p 4.08 0.43 -‐ -‐ FUT4 Fucosyltransferase 4 (Alpha (1,3) Fucosyltransferase, Myeloid-‐Specific)

Catalyses the synthesis of the non-‐sialylated antigen, Lewis x Acute ischemic stroke

SH2B3 SH2B Adaptor Protein 3 Involved in a range of signaling activities by growth factor and cytokine receptors Pancreatic cancer

ZMPSTE24 Zinc Metallopeptidase STE24 Proteolytically removes the C-‐terminal three residues of farnesylated proteins Prostate cancer

hsa-‐miR-‐1915-‐3p 4.01 0.52 0.86 1.37 -‐ -‐ -‐ -‐hsa-‐miR-‐1225-‐3p 3.9 0.57 1.34 1.07 MTMR11 Myotubularin Related Protein 11 Probable pseudophosphatase Sickle cell disease

RBPMS RNA Binding Protein With Multiple Splicing Acts as a coactivator of transcriptional activity PheochromocytomasC1QTNF3 C1q And Tumor Necrosis Factor Related Protein 3 -‐

hsa-‐miR-‐1304-‐5p 3.9 0.58 -‐ -‐ -‐ -‐ -‐ Lung adenocarcinomahsa-‐miR-‐572 3.9 0.35 -‐ -‐ ONECUT1 One Cut Homeobox 1 Transcriptional activator Renal cell carcinoma

CIB2 Calcium And Integrin Binding Family Member 2 May play a role in calcium homeostasis and participate in calcium regulation Ovarian cancer

STIP1 Stress-‐Induced Phosphoprotein 1 Mediates the association of the molecular chaperones HSC70 and HSP90 Autism spectrum disorder

hsa-‐miR-‐519b-‐3p 3.8 0.32 -‐ -‐ PIGK Phosphatidylinositol Glycan Anchor Biosynthesis, Class K

Mediates GPI anchoring in the endoplasmic reticulum Laryngeal carcinoma

FAM13A1 Family With Sequence Similarity 13, Member A1 -‐CDK2 Cyclin-‐Dependent Kinase 2 Participate in cell cycle regulation

hsa-‐miR-‐30e-‐5p 3.8 0.7 1.09 1.23 -‐ -‐ Alzheimer's diseaseNon-‐small cell lung cancer

hsa-‐miR-‐575 3.79 0.76 -‐ -‐ GNPDA2 Glucosamine-‐6-‐Phosphate Deaminase 2 -‐ Leukaemia

FLNB Filamin B, Beta Connects cell membrane constituents to the actin cytoskeleton Systemic lupus erythematosus

RAB6IP1 RAB6 interacting protein 1 Promotes the exchange of GDP to GTPhsa-‐miR-‐548ac 3.77 0.51 -‐ -‐ -‐ -‐ -‐ Multiple sclerosishsa-‐miR-‐526b-‐5p 3.76 0.35 -‐ -‐ -‐ -‐ -‐ -‐hsa-‐miR-‐525-‐3p 3.72 0.45 -‐ -‐ EBF1 Early B-‐Cell Factor 1 Transcriptional activator Hepatocellular carcinoma

SACM1L SAC1 Suppressor Of Actin Mutations 1-‐Like (Yeast) Phosphoinositide phosphatase that hydrolyses PtdIns(3)P and PtdIns(4)P

SIRT3 Sirtuin 3 Activates or deactivates mitochondrial target proteins by deacetylating key lysine residues

hsa-‐miR-‐296-‐3p 3.68 0.23 0.71 0.54 CTDSPL CTD (Carboxy-‐Terminal Domain, RNA Polymerase II, Polypeptide A) Small Phosphatase-‐Like

Negatively regulates RNA polymerase II transcription Preeclampsia

HMGN4 High Mobility Group Nucleosomal Binding Domain 4 -‐ Prostate cancer

POSTN Periostin, Osteoblast Specific Factor Induces cell attachment and spreading and plays a role in cell adhesion

hsa-‐miR-‐765 3.68 0.51 1.01 0.66 RRH Retinal Pigment Epithelium-‐Derived Rhodopsin Homolog May play a role in rpe physiology Stroke

ADH5 Alcohol Dehydrogenase 5 (Class III), Chi Polypeptide Catalyses the oxidation of long-‐chain primary alcohols Coronary artery disease

EFNB3 Ephrin-‐B3 May play a role in constraining the orientation of longitudinally projecting axons Prostate cancer

hsa-‐miR-‐519e-‐3p 3.67 0.6 -‐ -‐ -‐ -‐ -‐ -‐

hsa-‐miR-‐576-‐5p 3.67 0.33 -‐ -‐ CDH11 Cadherin 11, Type 2, OB-‐Cadherin (Osteoblast) Imparting to cells the ability to adhere in a homophilic manner Glioblastoma

NAMPT Nicotinamide PhosphoribosyltransferaseCatalyses the condensation of nicotinamide with 5-‐phosphoribosyl-‐1-‐pyrophosphate to yield

nicotinamide mononucleotideSystemic lupus erythematosus

ABI2 Abl-‐Interactor 2 May act in regulation of cell growth and transformation

hsa-‐miR-‐601 3.6 0.85 -‐ -‐ CTPS CTP Synthase 1 Catalyses the ATP-‐dependent amination of UTP to CTP Bladder cancer

FLT4 Fms-‐Related Tyrosine Kinase 4Promotes proliferation, survival and migration of

endothelial cells, and regulates angiogenic sprouting

Ovarian cancer

SIRT1 Sirtuin 1

Participates in the coordination of several separated cellular functions such as cell cycle,

response to DNA damage, metobolism, apoptosis and autophagy

Systemic lupus erythematosus

hsa-‐miR-‐371b-‐5p 3.6 0.71 1.22 1.29 MBNL2 Muscleblind-‐Like Splicing Regulator 2 Mediates pre-‐mRNA alternative splicing regulation -‐

RCAN2 Regulator Of Calcineurin 2 Inhibits calcineurin-‐dependent transcriptional responses

SRRM1 Serine/Arginine Repetitive Matrix 1 Involved in numerous pre-‐mRNA processing events

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Table 6.1: Top 30 miRs that are overexpressed in pSS patients vs SLE from Nanostring Screening.

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Appendix B

Reduced miRs pSS Fold SLE Fold pSS PBMCs pSS CECs Predicted Gene Targets Gene Description Roles Disease Associationhsa-‐miR-‐125b-‐5p 0.03 0.77 0.56 0.91 -‐ -‐ -‐ Breast cancerhsa-‐miR-‐16-‐5p 0.06 2.32 1.13 1.36 -‐ -‐ -‐ Preeclampsiahsa-‐miR-‐150-‐5p 0.17 0.76 1.31 1.13 -‐ -‐ -‐ Coronary heart disease

Myasthenia gravishsa-‐miR-‐342-‐3p 0.2 0.62 0.68 1.39 BTN2A1 Butyrophilin, Subfamily 2, Member A1 -‐ Non-‐small cell lung cancer

EDA Ectodysplasin A Involved in epithelial-‐mesenchymal signaling during morphogenesis of ectodermal organs Hepatocellular carcinoma

ID4 Inhibitor Of DNA Binding 4 Act as TSs but lack DNA binding activity Acute myeloid leukaemia

hsa-‐miR-‐146b-‐5p 0.24 0.42 0.81 1.47 NOVA1 Neuro-‐Oncological Ventral Antigen 1 May regulate RNA splicing or metabolism in a specific subset of developing neurons Atrial fibrosis

STRBP Spermatid Perinuclear RNA Binding Protein Plays a role in regulation of cell growth Thyroid carcinomaFBXL10 F-‐Box And Leucine-‐Rich Repeat Protein 10 Inhibits cell growth and proliferation

hsa-‐miR-‐30b-‐5p 0.34 1.21 0.68 1.08 -‐ -‐ -‐ Parkinson's diseasehsa-‐miR-‐32-‐5p 0.34 1.17 -‐ -‐ -‐ -‐ -‐ Coronary heart diseasehsa-‐miR-‐126-‐3p 0.35 6.15 -‐ -‐ -‐ -‐ -‐ Kaposi's sarcomahsa-‐miR-‐132-‐3p 0.36 0.17 0.25 1.39 -‐ -‐ -‐ Alzheimer's diseasehsa-‐miR-‐194-‐5p 0.42 0.68 0.9 1.2 -‐ -‐ -‐ Barrett's oesophagushsa-‐miR-‐15a-‐5p 0.42 1.02 -‐ -‐ -‐ -‐ -‐ Epilepsyhsa-‐miR-‐221-‐3p 0.46 2.83 1.01 1.36 -‐ -‐ -‐ Major depressive disorderhsa-‐miR-‐144-‐3p 0.47 1.18 -‐ -‐ -‐ -‐ -‐ Laryngeal carcinomahsa-‐miR-‐29b-‐3p 0.48 0.36 0.64 1.06 -‐ -‐ -‐ Chronic hepatitis Bhsa-‐miR-‐191-‐5p 0.49 1.77 0.87 0.96 -‐ -‐ -‐ Acute myocardial infarction

Alzheimer's disease

hsa-‐miR-‐142-‐3p 0.54 1.33 -‐ -‐ MAP3K7IP2 Mitogen-‐activated protein kinase kinase kinase 7-‐interacting protein 2 Serves as an adaptor linking MAP3K7 and TRAF6 Multiple sclerosis

DIRC2 Disrupted In Renal Carcinoma 2 Electrogenic metabolite transporter Systemic lupus erythematosus

ATF7IP Activating Transcription Factor 7 Interacting Protein Modulates transcription regulation and chromatin formation

hsa-‐miR-‐26b-‐5p 0.54 1.08 0.82 0.87 -‐ -‐ -‐ Primary biliary cirrhosis

hsa-‐miR-‐125a-‐5p 0.56 0.28 0.45 0.96 RBM7 RNA Binding Motif Protein 7 Possibly involved in germ cell RNA processing and meiosis Non-‐small cell lung cancer

TGOLN2 Trans-‐Golgi Network Protein 2 May be involved in regulating membrane traffic to and from trans-‐Golgi network Hepatocellular carcinoma

CPSF6 Cleavage And Polyadenylation Specific Factor 6, 68kDa Plays a key role in pre-‐mRNA 3'-‐processinghsa-‐miR-‐19b-‐3p 0.56 2.22 0.82 1.43 -‐ -‐ -‐ Preeclampsia

hsa-‐miR-‐98 0.58 1.74 -‐ -‐ UHRF2 Ubiquitin-‐Like With PHD And Ring Finger Domains 2, E3 Ubiquitin Protein Ligase

May contribute to a tighter epigenetic control of gene expression in differentiated cells Lung squamous cell carcinoma

LRIG3 Leucine-‐Rich Repeats And Immunoglobulin-‐Like Domains 3

May play a role in craniofacial and inner ear morphogenesis during embryonic development Hypoxia

RDH10 Retinol Dehydrogenase 10 (All-‐Trans) Converts all-‐trans-‐retinol to all-‐trans-‐retinal Juvenile angiofibromahsa-‐miR-‐19a-‐3p 0.6 2.93 0.82 1.56 -‐ -‐ -‐ Colorectal adenocarcinomahsa-‐miR-‐642b-‐3p 0.61 0.34 0.66 1.51 -‐ -‐ -‐ -‐hsa-‐miR-‐451a 0.61 1.93 -‐ -‐ -‐ -‐ -‐ Major depressive disorderhsa-‐miR-‐26a-‐5p 0.62 1.81 -‐ -‐ -‐ -‐ -‐ Colorectal cancer

hsa-‐miR-‐190b 0.64 0.53 -‐ -‐ EDIL3 EGF-‐Like Repeats And Discoidin I-‐Like Domains 3 Mediates angiogenesis and may be important in vessel wall remodeling and development Hepatocellular carcinoma

ARPC5 Actin Related Protein 2/3 Complex, Subunit 5, 16kDa Control of actin polymerisation in cells Lung adenocarcinomaACTR3 ARP3 Actin-‐Related Protein 3 Homolog (Yeast) Regulates actin polymerisation

hsa-‐miR-‐99b-‐5p 0.65 0.43 0.17 0.99 -‐ -‐ -‐ Fibromyalgiahsa-‐miR-‐148b-‐3p 0.65 2.1 0.97 1.24 -‐ -‐ -‐ Bladder cancerhsa-‐miR-‐25-‐3p 0.66 1.37 0.74 1.21 -‐ -‐ -‐ Macular degenerationhsa-‐miR-‐9-‐5p 0.66 0.24 -‐ -‐ -‐ -‐ -‐ Parkinson's diseasehsa-‐miR-‐2682-‐5p 0.67 0.5 -‐ -‐ -‐ -‐ -‐ -‐

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Table 6.2: Top 30 miRs that are reduced in pSS patients versus SLE from Nanostring Screening.

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