Oxidative Stress in Aging and Human Diseases - Exploring the Mechanisms

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Wei Cao, PhD

[email protected]

Oxidative Stress in Aging and Human Diseases – Exploring the Mechanisms

Oxidative stress and aging

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Agenda

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Oxidative stress in human diseases


Application example and solutions

Long non-coding RNAs (lncRNAs) in oxidative stress and aging

Summary and questions


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Oxidative stress and human health

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Oxidative stress damages DNA, proteins, lipids and carbohydrates and is related to the pathogenesis of different diseases, such as:

• Cancer

• Neurodegenerative diseases

• Cardiovascular diseases

• Obesity

• Atherosclerosis

• Diabetes

• Asthma

• Hepatic diseases

• Eye disease

• Dermatitis

• Pneumonia

• Aging

Pandey, K.B. and Rizvi, S.I. (2010) Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxid Med Cellular Longev. 3(1), 2-12


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Reactive oxygen species (ROS) and antioxidant defense mechanisms

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• ROS have a double role: they mediate both physiologic events and cellular damage

• Antioxidants neutralize ROS/RNS by accepting or donating electrons

◦ Endogenous enzymatic antioxidants: Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR)

◦ Non-enzymatic antioxidants: Vitamin C, uric acid, albumin, bilirubin, vitamin E (α-tocopherol), β-carotene and flavonoids

◦ Primary antioxidants: Terminate free-radical chain reactions by donating hydrogen or electrons to free radicals and converting them to more stable products

◦ Secondary antioxidants: Oxygen scavengers or chelating agents

MitochondriaPeroxisomesCytochrome P450

Exogenous sources

H2O , O2 , R-OH

STABLE PRODUCTS


ANTIOXIDANT FREE RADICAL


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Markers of oxidative stress and ROS

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• The uncontrolled production of ROS/RNS plays crucial roles in the onset of human diseases, so it is necessary to measure the level of oxidative stress

• Oxidative stress can be quantified in vivo through the measurement of markers which are related to ROS generation or molecules modified by interactions with ROS

Processes Markers of oxidative stress and ROS

Lipid peroxidation Malondialdehyde (MDA), F2-isoprostanes, oxidized low-density lipoproteins (LDL), oxidized LDL antibodies, advanced lipid oxidation products, acrolein, 4-hydroxynonenal

Protein oxidation Thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products, advanced glycation end-products (AGEs), disulfite formation

Carbohydrate oxidation Carbonyl formation, 3-nitrotyrosine

Nucleic acid oxidation Reactive aldehydes, reduced sugar (ascorbate, ribose, etc.), 8-oxy-2-deoxyguanosine

Serum antioxidant capacity Activity of antioxidant enzymes such as CAT, GPx, SOD

Regulators of gene expression miRNAs (such as miR-126), lncRNAs

Review article: “Today’s Oxidative Stress Biomarker” Vascular Pharmacology, 2015, 74:23



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Nrf2-ARE mediated oxidative stress response

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• Nrf2: Nuclear factor-erythroid 2-related factor is a transcription factor that binds to AREs (antioxidant responsive elements) to activate transcription

• Keap1: Kelch-like ECH-associated protein 1

• Normal conditions: Inactive Nrf2 is retained in the cytoplasm by association with Keap1

• Oxidative conditions: Nrf2 is phosphorylated, translocated to the nucleus, where it binds AREs and activates detoxifying enzymes and antioxidant enzymes

• Receptor modulation: PKC, PI3K and MAPK pathways modulate Nrf2 phosphorylation and translocation

Buendia, I. et al. (2016) Nrf2–ARE pathway: an emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacology & Therapeutics 157, 84–104

ARE


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Increased oxidative stress is linked to aging

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• Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death

• Increased oxidative stress has been linked to aging

Lopez-Otin, C. et al. (2013) The hallmarks of aging. Cell 153, 1194-1217Jones, D.P. (2015) Redox theory of aging. Redox Biology 5, 71-79

Giorgio, M. (2015) Oxidative stress and the unfulfilled promises of antioxidant agents. ecancer9, 556

The 9 hallmarks of aging

◦ Telomere attrition

◦ Cellular senescence

◦ Mitochondrial dysfunction

◦ Stem cell exhaustion

◦ Genomic instability

◦ Epigenetic alterations

◦ Loss of proteostasis

◦ Altered intercellular communication

◦ Deregulated nutrient sensing

◦ The redox theory of aging (also known as the free radical theory of aging) was proposed by Denham Harman in 1956

◦ Redox theory explains 9 hallmarks of aging

◦ Oxidative stress increases with increasing age. This condition leads to accumulation of oxidation products of lipids, nucleic acids, proteins and carbohydrates ultimately causing cellular dysfunction and making the body prone to external deleterious agents

• However, several experimental models of antioxidant manipulation have failed to affect the lifespan

• Moreover, antioxidant supplementation clinical trials have been disappointing


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Mitochondrial oxidative stress and aging

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Dai, D.F. et al. (2014) Mitochondrial oxidative stress in aging and healthspan. Longev. Healthspan 3, 6

The mitochondrial theory of aging – a revised theory• Specifies that mitochondria are both the primary sources of ROS and

the primary targets of ROS damage; therefore, mitochondrial ROS plays a central role in aging

• Growing experimental evidence shows the beneficial effects of mitochondrial-targeted antioxidants in aging and healthspan

Led to a focus on developing and refining drugs to specifically target ROS in the mitochondria of cells


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Mitochondrial oxidative stress and aging

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Dai, D.F. et al. (2014) Mitochondrial oxidative stress in aging and healthspan. Longev. Healthspan 3, 6


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Oxidative stress is linked to inflammation

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Fischer, R. and Maier, O. 2015. Interrelation of oxidative stress and inflammation in neurodegenerative disease: role of TNF. Oxid Med Cellular Longev.

• Oxidative stress promotes inflammation

• Usually, mediators of oxidative stress and inflammation are in balance with detoxifying and anti-inflammatory molecules

• During disease, this balance is shifted towards oxidative stress and pro-inflammatory sites, leading to DNA, protein and cell damage, inflammation and, finally, cell death

NF B links ROS/RNS 𝜅production and the induction of pro-inflammatory cytokines


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Aging is accompanied by inflammation

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Cannizzo, E.S. et al. 2011. Oxidative stress, inflamm-aging and immunosenescence” J Proteomics. 74(11), 2313

• Aging is accompanied by two to four-fold increases in plasma/serum levels of inflammatory mediators such as cytokines and acute phase proteins

• Stress, declining production and function of sex hormones, genetic factors, increased amount of fat tissue, impaired immune function, infection and smoking/pollution all contribute to systemic chronic low-grade inflammation in the elderly

• Aging is characterized as a systemic, chronic, low-grade inflammation. This is called “inflamm-aging.” Inflammaging is a predictor of fragility. TNF-α and IL-6 levels are biomarkers of frailty.

• Two major pathways: oxidative stress promotes inflammatory responses:

◦ Toll like receptors (TLRs)

◦ Nalp-3 inflammasomes

Aging

Inflammation

ObservationExperiment


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NF- B pathway in aging process𝜅

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https://www.qiagen.com/us/shop/genes-and-pathways/pathway-details/?pwid=315

• NF- B is the master regulator of the 𝜅inflammatory process

• NF- B over-activation is one of the 𝜅transcriptional signatures of aging

• Mechanism of NF- B over-activation: 𝜅◦ Strongly enhanced activity of IKK and three

MAPKs (ERK, P38 and JNK) during the aging process

◦ Increased reactive oxygen species (ROS) production while aging

• Inhibition of NF- B prevents aging 𝜅associated features in mouse models


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Aging and the telomere connection through inflammation

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Zhang, J. et al. 2015. Ageing and the telomere connection: an intimate relationship with inflammation. Ageing Res Rev. 25, 55–69

The interplay between inflammation and telomeres/telomerase in aging process• Mitochondria play a key role in initiating inflammatory pathways and mitochondrial dysfunction

drives the aging process

• Telomere shortening leads to accelerated ageing and increases risk of age-related diseases

• Inflammatory molecules are associated with telomere dysfunction in various diseases

• Some proteins have dual functions in both inflammation and telomere maintenance


Regulates TERT expression

NF-B𝜅

Wnt

PAPR

TERT

RAP1

Regulates TERT expression

Involved in DNA damage response at telomeres

Telomere lengthening and maintenance

Shelterin complex proteinTelomere stability

Transcription of pro-inflammatory genes

Expressed in immune cells

Stimulate expression of pro-inflammatory genes

Involved in NF- B 𝜅signaling pathway

Modulates NF- B 𝜅dependent transcriptions

Preserves proliferative capacity of immune cells

In Inflammation In TelomeresProtein

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Experimental Model:

Knockout of the nfkb1 subunit of the transcription factor NF- B to induce chronic inflammation𝜅qPCR assays to profile: • Targets of NF- B signaling𝜅• Genes involved oxidative stress

Application: inflammation causes aging

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Download the article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090717/pdf/ncomms5172.pdf

• Telomere dysfunction• Cell senescence • Tissue regeneration• DNA damage• ROS production• COX-2 expression

nfκb1-/- mice

Aging


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• Experimental model: knockout of the nfkb1 subunit of the transcription factor NF-kB to induce chronic inflammation

• qPCR arrays: ◦ NFkB Signaling Targets RT2 Profiler PCR Array◦ Oxidative Stress RT2 Profiler PCR Array


18Oxidative stress and aging


• Telomere dysfunction• Cell senescence • Tissue regeneration• DNA damage• ROS production• COX-2 expression

nfκb1-/- mice

Aging

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• Use QIAGEN RNeasy Mini Kit to isolate total RNA

• Use RT2 First Strand Kit for cDNA conversion◦ Integrated DNase step◦ Proprietary spike in RNA◦ Priming with both oligo-dTs as well as random

hexamers

• Use RT2 SYBR Green master mix for qPCR with RT2 Profiler PCR Arrays

◦ NFkB Signaling Targets RT2 Profiler PCR Array◦ Oxidative Stress RT2 Profiler PCR Array

• Go to QIAGEN’s Data Analysis Center to analyze gene expression data

Separatebeads

Experimental setupSamples: tissues from wild type & nfκb1-/- mice of the same age and late generation terc-/- mice



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Oxidative Stress RT2 Profiler PCR Array – 84 genes• Human, mouse, rat, etc. up to 14 species

• Antioxidants:◦ Glutathione peroxidases (GPx): GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSTP1,

GSTZ1◦ Peroxiredoxins (TPx): PRDX1, PRDX2, PRDX3, PRDX4, PRDX5, PRDX6 (AOP2)◦ Other peroxidases: CAT, CYBB, CYGB, DUOX1, DUOX2, EPX, LPO, MGST3, MPO, PTGS1,

PTGS2 (COX2), PXDN, TPO, TTN◦ Other antioxidants: ALB, APOE, GSR, MT3, SELS, SOD1, SOD3, SRXN1, TXNRD1, TXNRD2

• Genes involved in ROS metabolism:◦ Superoxide dismutases (SOD): SOD1, SOD2, SOD3◦ Other genes Involved in superoxide metabolism: ALOX12, CCS, DUOX1, DUOX2, GTF2I, MT3,

NCF1, NCF2, NOS2 (iNOS), NOX4, NOX5, PREX1, UCP2◦ Other genes involved in ROS metabolism: AOX1, BNIP3, EPHX2, MPV17, SFTPD◦ Oxidative stress responsive genes: APOE, ATOX1, CAT, CCL5 (RANTES), CYGB, DHCR24,

DUOX1, DUOX2, DUSP1 (PTPN16), EPX, FOXM1, FTH1, GCLC, GCLM, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSR, GSS, HMOX1, HSPA1A, KRT1, LPO, MBL2, MPO, MSRA, NQO1, NUDT1, OXR1, OXSR1, PDLIM1, PNKP, PRDX2, PRDX5, PRDX6 (AOP2), PRNP, RNF7, SCARA3, SELS, SEPP1, SIRT2, SOD1, SOD2, SQSTM1, SRXN1, STK25, TPO, TTN, TXN, TXNRD1, TXNRD2

• Oxygen transporters: CYGB, MB

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Oxidative Stress RT2 Profiler PCR Array


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Cytokines & chemokines:Ccl12 (MCP-5, Scya12), Ccl22 (MDC), Ccl5 (RANTES), Csf1 (Mcsf), Csf2 (GMCSF), Csf3 (Gcsf), Cxcl1 (Gro1), Cxcl10(INP10), Cxcl3, Cxcl9 (Mig), Fasl (Tnfsf6), Ifnb1, Ifng, Il12b, Il15, Il1a, Il1b, Il1rn, Il2, Il4, Il6, Lta (Tnfb), Ltb, Tnf, Tnfsf10(Trail)Inflammation:Acute Inflammation: C3, C4a, Ccl5 (RANTES), Cfb (Bf), F3, F8, Il1a, Il1b, Il6, Ins2, Stat3, Stat5b.Other Pro-Inflammatory Genes: Agt, Akt1, Ccl12 (MCP-5, Scya12), Ccl22 (MDC), Ccr5, Cd40 (Tnfrsf5), Cxcl1 (Gro1),Cxcl10(INP10), Cxcl3, Cxcl9 (Mig), Il15, Il1rn, Il2, Il2ra (CD25), Myd88, Ptgs2 (COX2), Sele, Selp, Tnf, Tnfrsf1b.Apoptosis:Agt, Birc2 (cIAP1, cIAP2), Cd74, Egfr, Fasl (Tnfsf6), Gadd45b, Ifnb1, Ifng, Il12b, Il2ra (CD25), Il4, Ins2, Lta (Tnfb), Map2k6(Mek6, Mkk6), Mitf, Mmp9, Nqo1, Nr4a2 (Nurr1), Ptgs2 (COX2), Stat1, Tnfrsf1b, Tnfsf10 (Trail), Trp53 (p53), Traf2.Anti-apoptotic:Adm, Akt1, Bcl2a1a (Bfl-1, A1), Bcl2l1 (Bcl-XL), Birc3 (cIAP1, cIAP2), Ccl12 (MCP-5, Scya12), Cdkn1a (p21Cip1, Waf1),Csf2 (GMCSF), F3, Fas (Tnfrsf6), Il1a, Il1b, Il2, Il6, Myd88, Nfkbia (Iκbα, Mad3), Sod2, Stat5b, Tnf, Xiap (Birc4).Immune response:Innate Immunity: C3, C4a, Ccl12 (MCP-5, Scya12), Cfb (Bf), Ifnb1, Il12b, Il6, Ins2, Myd88, Nfkbia (Iκbα, Mad3), Stat5b, Tnf.Adaptive Immunity: C3, C4a, Ccl12 (MCP-5, Scya12), Cd40 (Tnfrsf5), Cd74, Cd80, Icam1, Ifng, Il12b, Il1b, Il2, Il4, Traf2.Other Immune Response Genes: Cd83, Fas (Tnfrsf6), Fasl (Tnfsf6), Il1r2, Lta (Tnfb), Ltb, Tnfsf10 (Trail).Type I interferon responsive genes:Adm, Ccl12 (MCP-5, Scya12), Ccl5 (RANTES), Cd80, Cdkn1a (p21Cip1, Waf1), Cfb (Bf), Cxcl10 (INP10), Cxcl9 (Mig), Il15,Il1rn, Irf1, Myd88, Ncoa3, Stat1, Tnfsf10 (Trail).Differentiation & development:Lymphoid Differentiation & Development: Cd80, Cd74, Cd83, Il12b, Il15, Il2, Il2ra (CD25), Il4, Irf1, Stat5b, Trp53 (p53),Vcam1.Myeloid Differentiation & Development: Ccl5 (RANTES), Csf1 (Mcsf), Csf2 (GMCSF), Csf2rb, Csf3 (Gcsf), Il4, Mitf, Mmp9,Nfkbia (Iκbα, Mad3), Stat5b, Tnf.Nervous System Differentiation & Development: Agt, Aldh3a2, Cxcl1 (Gro1), Egfr, Egr2, Ifng, Nr4a2 (Nurr1), Snap25, Sod2, Stat3, Trp53Stress responses:Adm, Akt1, Bcl2l1 (Bcl-XL), Birc2 (cIAP1, cIAP2), Ccnd1, Cdkn1a (p21Cip1, Waf1), Gadd45b, Ifng, Il1a, Il1b, Map2k6 (Mek6, Mkk6), Nqo1, Pdgfb, Plau (uPA), Sod2, Tnf, Trp53 (p53), Xiap (Birc4).NFκB signaling:Transcription Factors: Nfkb1, Nfkb2, Nfkbia (Iκbα, Mad3), Relb, Stat1.Regulation of NFκB Signaling: Birc2 (cIAP1, cIAP2), Cd40 (Tnfrsf5), Fasl (Tnfsf6), Il1b, Myd88, Rel, Rela, Tnf, Tnfsf10(Trail).Transcription factors:NFκB Signaling: Nfkb1, Nfkb2, Rel, Rela, Relb.Other Transcription Factors: Egr2, Irf1, Mitf, Myc, Nr4a2 (Nurr1), Stat1, Stat3, Stat5b, Trp53 (p53).

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NFkB Signaling Targets RT2 Profiler PCR Array


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=20293

























































































































































































































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Conclusions:• Systemic chronic inflammation can accelerate aging via ROS-mediated exacerbation of

telomere dysfunction and cell senescence• There exists a positive feedback loop system between telomere dysfunction, senescence-

associated ROS production and pro-inflammatory signaling that induces and stabilizes senescence in vivo, which in turn limits the regenerative capacity of tissues

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RT2 Profiler PCR Arrays System

• Profile 84 different genes on one array

• Appropriate controls for data normalization, sample quality and reaction performance

◦ Five housekeeping genes◦ A Genomic DNA

contamination control (GDC)◦ Three reverse transcription

controls (RTCs)◦ Three positive PCR controls

(PPCs)

• Allows you to focus on your questions and papers

Pathway or disease-focused gene expression profiling


Extract total RNA

Make cDNA

Mix with master mix Load plate Run qPCR Analyze

data

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More than 170 arrays covering up to 13 species• Aging• DNA damage• Oxidative stress• Telomeres & telomerase• Cellular senescence• p53 signaling• NFκB signaling• NFκB targets• Cell cycle• Apoptosis• Autophagy• Growth factors• mTOR signaling• AMPK signaling• Insulin resistance• Stem cells• Cancer stem cells

• Inflammatory cytokines and receptors• Cancer inflammation and immunity

crosstalk• Chemokines and receptors• Common cytokines• Cytokines and chemokines• TNF signaling pathway• Antiviral response• Inflammatory response & autoimmunity• Toll-like receptors (TLRs)• Innate and adaptive immune responses• Inflammasomes• IL-6/STAT3 signaling• T helper cell differentiation• Th1 and Th2 responses• Th17 response• Interferons and receptors• MAPK signaling• TGFβ/BMP signaling


RT2 Profiler PCR Arrays and Assays

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Aging RT2 Profiler PCR Array

• Genomic instability: BUB1B, MRPL43, POLRMT, TFAM, TFB1M, TFB2M, ZMPSTE24• Telomere attrition: POT1, RAP1A, TERF1, TERF2, TINF2, TPP1• Mitochondrial dysfunction: MRPL43, NDUFB11, POLRMT, SIRT1, SIRT3, SIRT6, TFAM, TFB1M, TFB2M• Proteostasis: ARL6IP6, BUB1B, FOXO1, HSF1, HSF1, JAKMIP3, RNF144B, SIRT1, TXNIP, VPS13C• Laminopathies: LMNA, LMNB1, LMNB2, ZMPSTE24• Neurodegeneration & synaptic transmission: CALB1, GFAP, MBP, SCN2B, SNAP23• Epigenetic alterations: ARID1A, SIRT1, SIRT3, SIRT6• DNA binding: ARID1A, ELP3, EP300, FBXL16, ZBTB10, ZFR, ZNF25• RNA binding: ELAVL1, LSM5, ZFR• Inflammatory response: ANGEL2, ANXA3, ANXA5, C1QA, C1QB, C1QC, C1S, C3, C3AR1, C5AR1,

CCR1, CD14, CD163, CFH, CX3CL1, CXCL16, FCER1G, FCGBP, FCGR1A, FCGR2A, FCGR3B, GFAP, LTF, LYZ, MBP, PANX1, S100A8, S100A9, TMEM135, TMEM33, TLR2, TLR4, TOLLIP

• Apoptosis: CASP1, CLU, EP300, PDCD6, TOLLIP• Cellular senescence: CDKN1C, VWA5A, WRN• Cell cycle: BUB1B, CDKN1C• Cytoskeleton: COL1A1, COL3A1, EML1• Oxidative stress: EP300, GSTA1• Transcriptional regulation: ARID1A, EP300, FOXO1, HSF1, PHF3, SMAD2

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Long non-coding RNAs (lncRNAs): new players in aging

Grammatikakis, I. et al. (2014) Long noncoding RNAs (lncRNAs) and the molecular hallmarks of aging. Aging 6(12), 992-1009

The roles of lncRNAs in aging:• Modulate telomere length• Control epigenetic alterations in

aging and senescence• Associated with proteostasis,

including autophagy and protein synthesis, trafficking, assembly and degradation

• Modulate stem cell homeostasis• Involved in cell cycle regulation• Regulate intercellular

communication

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• lncRNAs are non-protein-coding transcripts longer than 200 nucleotides

• Most lncRNAs are localized in the nucleus, but some are found in the cytoplasm

• Many lncRNAs are molecularly indistinguishable from mRNAs and share many features of mRNAs

• Although some lncRNAs (e.g. MALAT1) are highly abundant transcripts, many lncRNAs are less so. But low transcription levels do not necessarily reflect lack of functionality

• lncRNAs may or may not contain a poly-A tail (mRNAs have a poly-A tail)

• lncRNAs are typically less conserved across species and often show low expression levels and high tissue specificity

• Expression of lncRNAs is generally lower than that of mRNAs and sensitive method such as qPCR are needed to detect and quantify them

Schwarzenbach, H. et. al. (2013) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426 Rönnau, C.G.H. (2014) Noncoding RNAs as novel biomarkers in prostate cancer. Biomed. Res. Int. 591703

What are lncRNAs?


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lncRNA classification and subgroup

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Wu P. et al. (2013) Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases. Brain Research Bulletin 97, 69

• lncRNAs can exceed 100,000 nucleotides in length and cover a wide range of gene positions

• lncRNAs can be divided into three general categories:◦ Transcribed relative to host protein-coding genes◦ Transcribed from gene regulatory regions◦ Transcribed from specific chromosomal regions

Intergenic

Intronic

Exonic

Overlapping

Sense

Antisense

Classifying lncRNAs based on their relative position to PCG (protein-coding genes)


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The diverse functions of lncRNAs


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lncRNAs in oxidative stress and aging-related disorders

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• Under oxidative stress◦ Expression of lncRNAs change during oxidative stress◦ lncRNAs are induced during various types of stress, including genotoxic stress, oxidative

stress and endoplasmic reticulum (ER) stress◦ They are involved in the heat shock response, DNA damage response and hypoxia

• Aging process◦ lncRNAs are deregulated in aging-related diseases and serve as potential molecular markers

and therapeutic targets



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Application: oxidative stress-induced changes in lncRNAs

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Study: The effect of oxidative stress on the transcriptome of human fibroblasts

Methods:• Cell culture: MRC5 and BJ fibroblasts• Oxidative conditions: treated cells for 30

minutes or 2 hours with 0.2 mM H2O2

• Isolated total RNA from treated cells

Findings:• Detected that 14,639 (68.7%) out of 21,311 lncRNAs were up-regulated• Four lncRNAs groups are prominent:

◦ dncRNAs (65.1%)◦ antisense overlapping lncRNAs (65.9%); ◦ terminal-associated lncRNAs transcribed at the same direction as their protein-coding genes pairs (70.7%)◦ promoter-associated antisense lncRNAs (75.9%)

Stress-induced lncRNAs are an integral part of the core transcriptional response to environmental stress and may facilitate cellular adaptation to stress

Analysis: • Used RNA-seq, ChIP-seq and microarrays to analyze the genomic response: upstream, coding and

downstream• RT-qPCR used to verify results



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Current lncRNA quantification approaches

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• RNA-seq (whole transcriptome sequencing): discover new RNAs and splicing variants

• Microarrays: use data analysis approaches to identify lncRNAs

• Real-time PCR based approaches: sensitive and quantitative for low-expressingRNAs and small gene changes; gold standard for gene quantification; able to use pre-amplification and WTA strategies (FFPE and single-cell analysis)

Test and verify your hypothesis with: RT2 lncRNA qPCR Assays or Custom PCR Arrays


miRNeasy andexoRNeasy

Serum/Plasma Kit

RT² lncRNAPreAMP PCR Kit

RT2 lncRNA PCR System

Free data analysis tool

Sample isolation Amplification qPCR Data analysis

& interpretation

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RT2 lncRNA qPCR system

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• lncRNA databases: In-house database at QIAGEN GeneGlobe, currently covering human GENCODE19, mouse GENCODE M2, RefSeq Release 65 and more than 40,000 human and 27,000 mouse lncRNA assays

• RT2 lncRNA assays: Laboratory-verified for optimal qPCR performance with high specificity, amplification efficiency and sensitivity

• RT2 lncRNA qPCR Arrays: Pathway or disease relevant lncRNA assays to facilitate comparative discovery in cancer and other research fields◦ RT2 lncFinder PCR Array (human and mouse)◦ RT2 lncRNA Cancer PathwayFinder Array (human and mouse)◦ RT2 lncRNA Inflammatory Response & Autoimmunity (human and mouse)◦ RT2 lncRNA Cell Development & Differentiation (human and mouse)

• Custom option: Flexible custom design from the lncRNA and qPCR databases allows profiling of mRNAs and lncRNAs simultanously

• Data analysis: Free on-line data analysis tools

https://www.qiagen.com/us/search/rt2-lncrna-pcr-arrays/


https://www.qiagen.com/us/search/rt2-lncrna-pcr-arrays/

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RT2 lncRNA qPCR Array – format and controls

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• Flexible layout and patented controls• Each 96-well plate has:

◦ 84 lncRNA-specific assays◦ Five reference genes (3 mRNAs and 2 lncRNAs)◦ A genomic DNA control (GDC)◦ Three reverse transcription control (RTC)◦ Three PCR controls (PPC)

384-well format(4x96)

RT2 Custom Array Builder: easy and flexible


96-well format

100-well ring

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Build your custom PCR array at: https://www.qiagen.com/myPCRarray

Integrate coding & noncoding RNA expression analysis

How to get started with the new custom array builder? Watch a movie:

https://attendee.gotowebinar.com/recording/6162050561736908290

Gene expression regulates biology

Integrate coding and noncoding RNA expression analysis

Select mRNA and lncRNA assays to build your custom PCR array


https://www.qiagen.com/myPCRarray

https://attendee.gotowebinar.com/recording/6162050561736908290

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. Whole genome• Illumina gene expression profiling• Illumina genotyping

. Pathway/focused panel• Mutation profiling• Methylation• PCR arrays• miRNA PCR arrays• NGS

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. Individual gene/locus• Mutation detection• Methylation• qPCR

. Sample preparation – DNA, RNA extraction and purification• Cells, tissue or biofluids• Fixed tissue• Small samples

Visit service: http://www.qiagen.com/products/catalog/services/

Contact: [email protected]


We provide service – send your samples to us and receive results

http://www.qiagen.com/products/catalog/services/

mailto:[email protected]

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Agenda

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Study oxidative stress and aging

• Cellular pathways require molecular signaling for their activity

◦ RT2 Profiler PCR Arrays

◦ RT2 lncRNA PCR Arrays

◦ RT2 Custom PCR Arrays: interaction of coding and non-coding RNAs

• QIAGEN offers Sample to Insight solutions

◦ Sample prep

◦ Real-time PCR assays

◦ Data analysis & interpretation

• Service Core

◦ Send us your samples and receive results


Choose QIAGEN and turn your hypotheses into actionable insights!

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Thank you for attending

Thank you for attending today’s webinar!

Contact QIAGENCall: 1-800-426-8157

Email: [email protected]

[email protected]

Wei Cao, [email protected]

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Oxidative Stress in Aging and Human Diseases - Exploring the Mechanisms

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Transcript of Oxidative Stress in Aging and Human Diseases - Exploring the Mechanisms