Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Series Part 4

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Functional analysis of miRNA

Martin Kreutz

[email protected]

Scientist, Product Development

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Webinar 4: Functional Analysis of miRNA 2

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Welcome to our four-part webinar series on miRNAs


Part 1: Biofluid miRNA profiling: from sample to biomarker

Part 2: Meeting the challenges of miRNA research

Part 3: Advanced miRNA expression analysis

Part 4: Functional analysis of miRNA

miRNA and its role in human disease

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miScript miRNA Target Protectors4

Agenda

miRNA background1

miScript miRNA Mimics2

miScript miRNA Inhibitors3

4

Controls5

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Changes in miRNA can be

correlated with gene expression

changes in development,

differentiation, signal transduction,

infection, aging and disease

After expression analysis of

miRNAs, functional studies are the

next step to understand the meaning

of the observed changes in

expression

As a single miRNA can have a large

group of potential targets. Functional

analysis is a valuable tool to

investigate the involvement of a

differentially regulated miRNA in the

process of interest

miRBase Entries

Webinar 4: Functional Analysis of miRNA

Why assess the function of microRNAs (miRNAs)?

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6Webinar 4: Functional Analysis of miRNA

Canonical pathway of miRNA biogenesis

Transcribed by RNA polymerase II as a long primary transcript (pri-miRNAs), which may contain more than one miRNA

In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by the RNase III Drosha

Pre-miRNAs are then exported to the cytosol by exportin 5

In the cytosol, the RNAse III Dicer processes these precursors to mature miRNAs

These miRNAs are incorporated in RISC

miRNAs with high homology to the target mRNA lead to mRNA cleavage

miRNAs with imperfect base pairing to the target mRNA lead to translational repression and / or mRNA degradation

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Mechanisms of gene silencing by miRNA:

Ribosome drop-off

Deadenylation and degradation

miRNA sequestration

Translational Repression

and/or

Transcript DegradationP Body Sequestration

Translational Repression

and/or

Transcript DegradationP Body Sequestration

Guo, H et.al., (2010) Nature. 466: 835-40, Bartel D.P., (2009) Cell 136; 215, Grimson A et.al., (2007) Mol Cell.27: 91-105.


How do miRNAs regulate gene expression?

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How do miRNAs interact with target mRNAs?


Basis of miRNA–mRNA interaction‏

Seed region: nucleotides 2–7 in 5′ region of miRNA

Most evolutionary conserved miRNA region

Most frequently complementary to target 3′‏-UTRs

Often sufficient to confer mRNA recognition

Beyond the seed region

end also contributes (extensive pairing is rare)‏3′

Some cases: central 11–12 continuous base pairs

Result of interaction

Suppression of gene expression

Rare cases: increase gene expression

References

Grimson, A., et al, Mol. Cell 2007, 27, 91-105

Image From Bartel, D.P., Cell 2009, 136, 215-233

Guo, H., et al, Nature 2010, 466, 835-840

Thomson, D.W., et al, Nucleic Acids Res 2011, 1-9

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How to determine miRNA–mRNA interactions


Prediction Algorithm Website

TargetScan http://www.targetscan.org/

Pictar http://pictar.mdc-berlin.de/

MicroCosm Targets http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/

DIANA http://diana.cslab.ece.ntua.gr/microT/

miRANDA http://www.microrna.org/microrna/home.do

TarBase (experimentally supported)

http://diana.cslab.ece.ntua.gr/tarbase/

:Target Prediction is based on‏

Bioinformatics

Seed region match

Position in 3′ UTR

Cross species conservation

Central sequence homology

Wet-lab research

Empirical evidence from microarrays

Reporter systems

:Pitfalls of using prediction algorithms‏

Large number of candidate mRNAs for a given

miRNA

May not incorporate all miRNA targeting

possibilities

Different algorithms produce different target lists

Potential for false positive rate of prediction

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Artificial manipulation of the normal level of a miRNA in a cell

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miRNA mimic and inhibitor experiment workflow

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Agenda

miRNA background1



12

Controls5

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miScript miRNA Mimics

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miScript miRNA Mimic principle

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‏ Features of miScript miRNA Mimics:

Double-stranded RNA oligonucleotide

Functional sequence is the same as the natural mature miRNA

Transfection results in inhibition comparable to the endogenous miRNA

Stable in culture for up to 72 hours


miScript miRNA Mimics mock endogenous miRNA activity

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‏ Analysis of miScript miRNA Mimic effects:

Reporter constructs (e.g. luciferase reporters)

Change in protein expression level

Change in mRNA expression in some cases

Downregulation by miRNA does not necessarily lead to degradation of the

target mRNA

Inhibition of endogenous HDAC4 using a miR-1 mimic

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Example of using a miScript miRNAMimic

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Target-dependent time course of effect

Changes in protein expression level

can often be measured after 48–72

hours

Can depend on different factors

Time course can differ dependent on

both miRNA and target

Stability and turnover of the

targets of the same miRNA might

differ

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Analysis of miScript miRNA Mimic effects

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Agenda

miRNA background1



18

Controls5

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miScript miRNA Inhibitors

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miScript miRNA Inhibitor principle

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‏ Features of miScript miRNA Inhibitors:

Single-stranded, chemically modified RNA oligonucleotide

Designed and modified to ensure efficient inhibition of endogenous miRNA

Transfection of inhibitor counteracts miRNA-induced silencing


miScript miRNA Inhibitors block endogenous miRNA activity

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miScript miRNA Inhibitors are effective after 96 hours

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miScript miRNA Inhibitor stability

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55 kD

28 kD Bcl2

TUBB

UTmiR-16

mimicAllStar

mimic

miR-15

Inhib.AllStar

Inhib.

miR-16

Inhib.

miR-15

& -16

Inhib.

‏ Analysis of miScript miRNA Inhibitor effects:

miRNA targets can be (and often are) regulated by more than one miRNA

Observing a miRNA inhibitor effect can require inhibiting more than one miRNA

BCL2 is regulated by miR-15a & miR-16

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Example of using a miScript miRNAInhibitor

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PDCD4

TUBB

miR-21

mimic

AllStar

mimic

miR-21

Inhib.

AllStar

Inhib.72 kD

55 kD

‏ Effects of miRNA mimic and inhibitor:

Transfection of miR-21 mimic leads to further reduction of endogenously

expressed PDCD4

Transfection of miR-21 mimic leads to an increase of PDCD4 protein

Regulation of PDCD4 by miR-21

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Example of using miScript miRNA mimics and inhibitors

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Agenda

miRNA background1



25

Controls5

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miScript miRNA Target Protectors

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miScript miRNA Target Protector principle

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‏ Features of miScript miRNA Target Protectors:

Single-stranded, chemically modified RNA oligonucleotide

Designed and modified to ensure efficient protection of specific mRNAs from

endogenous miRNAs

Transfection of target protector counteracts miRNA-induced silencing of a

single target

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miScript miRNA Target Protectors protect specific transcripts

Protection of a luc reporter against endogenously expressed miR-16

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‏ Analysis of miScript miRNA Target Protector effects:

Target protectors are designed against a single target site

If more than one miRNA targets the site, the target protector counteracts the

effect of both

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Protection of endogenously expressed targets

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miR-1 mimic was co-transfected in all samples except UT


Target protectors are designed against a single target site

Other targets affected by the same miRNA are not protected

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Target specificity of miScript miRNA Target Protectors

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Example: miR-9 causes downregulation of a gene that is important for cell

viability

After transfection of a miR-9 mimic, an increase of cell death is observed

miScript Target Protector miR-9 inhibits the downregulation by miR-9, allowing

the cell viability gene to be expressed

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Example of using a miScript miRNATarget Protector

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Target protectors in literature


Nakashima et al; Journal of Immunology 188, 2012.

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Nakashima et al; Journal of Immunology 188, 2012.

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Takeshita et al; International Journal of Oncology 41: 1653-1661. 2012

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Agenda

miRNA background1



36

Controls5

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Mimic

negative control

AllStars Negative Control siRNA

No homology to any mammalian gene

Validated for non-effects in microarray and phenotype assays

Validated for RISC entry

Mimic

positive control

Syn-hsa-miR-1 mimic

Not expressed under most culture conditions; only expressed in muscle cells

To check for optimal conditions by using miScript PCR miR-1

Inhibitor

negative control

miScript Inhibitor Negative Control

Target the sequence of miScript mimic negative control

Inhibitor

positive control

Anti-hsa-miR-1 inhibitor

To check for optimal conditions in combination with Syn-hsa-miR-1 mimic

Transfection

control

AllStars Hs Cell Death Control siRNA

Cell death = successful transfection

To run in every experiment


Controls for miRNA functional studies

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‏ Important aspects to consider:

Are both miRNA and target of interest expressed under the experimental

conditions?

What detection system is suitable?

How does the miRNA regulate the target (mRNA or protein level)

Could the target be regulated by other miRNAs?

Is the chosen cell line suitable for the expected time frame?

Does the miRNA affect cell viability?

miRNA functional analysis tips

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Where can I find the QIAGEN products discussed today?


www.qiagen.com

www.qiagen.com/GeneGlobe

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QIAGEN Sample to Insight Solutions for miRNA research

Sample PrepReal-time

PCR Assays

Data

AnalysisInterpretation

QIAcube

miRNeasy Mini

miRNeasy Micro

miRNeasy FFPE

miRNeasySerum / Plasma

ExoRNeasySerum / Plasma

Instruments

QIAgilityRotorGene QCompatibility with all Real-Time PCR instruments

miScript PCR System

miScript PreAMP

miScript Microfluidics

miScript PCR Arrays

miScript Primer Assays

GeneGlobe Data Analysis Center

Kits/Solutions

Ingenuity Pathway Analysis

miScript MimicsmiScript InhibitorsmiScript Target Protectors

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Thank you for attending today’s webinar!

Martin Kreutz

[email protected]

Contact QIAGEN

1-800-426-8157

[email protected]

Questions?

Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Series Part 4

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Transcript of Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Series Part 4