Basics of MicroRNA Study in Diabetes What is MicroRNA

8/21/2019 Basics of MicroRNA Study in Diabetes What is MicroRNA

1/24

Basics of microRNA study in

diabetes

Young-Kook Kim


2/24

~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?


3/24

MicroRNA biogenesis in animal


4/24

shRNA vector

siRNA duplex

siRNA or shRNA to the pathway


5/24

MicroRNA

Target

gene Function

Upstream

signaling

Downstreamsignaling

MicroRNAs function by target regulation


6/24

MicroRNA regulation of

insulin signaling and glucose homeostasis

Rottiers and Nr, NRMCB 13:239


7/24

Functional study of

microRNAs

MicroRNA-based

Expression profiling

Target gene-based

Screening Reporter, Mutagenesis

Target search bioinformatics


8/24

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


9/24

Guay et al, Translational Res 157:253


10/24

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


11/24

Normal Tissue

TumorTissue

with JieunYu in Han-KwangYang Lab

Testing with several companies- Agilent, Affymetrix, Invitrogen

Screening

- MicroRNAs increased in stomach cancer


12/24

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


13/24

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


14/24

Target prediction algorithm - TargetScan www.targetscan.org


15/24

Target prediction algorithm - TargetScan


16/24

Target gene verification Protein level? mRNA level?

Which cell lines?

What kind of (and how much) inhibitor?

3-UTR cloning and Luciferase assay


17/24

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


18/24

Mutation at the seed sequence

Direct regulation of target gene

mutagenesis


19/24

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


20/24

MicroRNAs in therapy miRNA inhibitor

(ex) miR-122

Biomarker

Exosome vesicle

Ago-associated miRNAs HDL-associated miRNAs

Circulating miRNAs as endocrine signals


21/24

Silencing of miR-122 in non-human

primates by LNA-antimiR

Elmen et al, Nature 452:896

LNA as microRNA inhibitor


22/24

Circulating microRNAs as potential

biomarkers for diagnosis and prognosis

Kosaka et al, Cancer Sci 101:2087


23/24

Circulating microRNAs associated with HDL

Rottiers and Nr, NRMCB 13:239


24/24

MicroRNA study in the future

More functional studies are required.

More studies are required for clinical application.

Basics of MicroRNA Study in Diabetes What is MicroRNA

Documents