Basics of MicroRNA Study in Diabetes What is MicroRNA
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Basics of microRNA study in
diabetes
Young-Kook Kim
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~22nt single-stranded RNAs Generated from endogenous stem-loop precursors
Often conserved among species
Abundant in animals and plants (100-1000 genes / species)
Function in a variety of regulatory pathways
(development, differentiation, proliferation, tumorigenesis, antiviral defense)
~22nt
What is microRNA?
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MicroRNA biogenesis in animal
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shRNA vector
siRNA duplex
siRNA or shRNA to the pathway
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MicroRNA
Target
gene Function
Upstream
signaling
Downstreamsignaling
MicroRNAs function by target regulation
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MicroRNA regulation of
insulin signaling and glucose homeostasis
Rottiers and Nr, NRMCB 13:239
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Functional study of
microRNAs
MicroRNA-based
Expression profiling
Target gene-based
Screening Reporter, Mutagenesis
Target search bioinformatics
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Discovering microRNA in diabetes Type I diabetes
Nonobese mouse
Streptozotocin-treated mouse
Type II diabetes
ob/ob mouse db/db mouse
Goto-Kakizaki rat
Patients
Blood sample
Tissue
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Guay et al, Translational Res 157:253
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How to measure the level of microRNA? Low throughput
Northern
Real Time PCR
High throughput
Microarray TaqMan Real Time PCR Array
High Throughput Sequencing
Chen et al, NAR 33:e179
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Normal Tissue
TumorTissue
with JieunYu in Han-KwangYang Lab
Testing with several companies- Agilent, Affymetrix, Invitrogen
Screening
- MicroRNAs increased in stomach cancer
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To find the target of microRNA Biochemical method is hard to set up
Ago-CLIP-Seq
MicroRNA over-expression / inhibition
Microarray or High-throughput sequencing
Hundreds of target candidates
Target prediction program Hundreds of target candidates
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Target gene prediction Target search through bioinformatics program
TargetScan
miRanda (miRBase) PicTar
RNAHybrid
Diana-microT miTarget
:
Intersect with
expression profiling data
miRNA
mimics
miRNA
mimics
Target
prediction
Target
predictionmiRNAinhibitormiRNAinhibitor
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Target prediction algorithm - TargetScan www.targetscan.org
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Target prediction algorithm - TargetScan
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Target gene verification Protein level? mRNA level?
Which cell lines?
What kind of (and how much) inhibitor?
3-UTR cloning and Luciferase assay
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miR-222~221 induces both mRNA degradation
and translational inhibition of p27 and p57.
(HeLa)
(HeLa)
(SNU-638)
Verification
- MicroRNA overexpression or inhibitionmiRNA mimic miRNA inhibitor
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Mutation at the seed sequence
Direct regulation of target gene
mutagenesis
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Apply to physiological system in each lab
(ex) miR-222-221 transgenic mouse
Physiological experiment
Stomach, adenoma
Duodenum, cancer
with HK Yangs & WH Kims lab
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MicroRNAs in therapy miRNA inhibitor
(ex) miR-122
Biomarker
Exosome vesicle
Ago-associated miRNAs HDL-associated miRNAs
Circulating miRNAs as endocrine signals
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Silencing of miR-122 in non-human
primates by LNA-antimiR
Elmen et al, Nature 452:896
LNA as microRNA inhibitor
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Circulating microRNAs as potential
biomarkers for diagnosis and prognosis
Kosaka et al, Cancer Sci 101:2087
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Circulating microRNAs associated with HDL
Rottiers and Nr, NRMCB 13:239
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MicroRNA study in the future
More functional studies are required.
More studies are required for clinical application.