microRNAsmicroRNAs

Small molecules, big functionsSmall molecules, big functions

Ali BazarganAli Bazargan

Brief historyBrief history

•The first described microRNA, lin-4 was cloned and characterised as a The first described microRNA, lin-4 was cloned and characterised as a translational repressor of developmental timing from Caenorhabditis. translational repressor of developmental timing from Caenorhabditis. elegans by Lee et al (1993) and Wightman et al (1993). elegans by Lee et al (1993) and Wightman et al (1993).

•The transcript of this gene was highly unusual as it was non-coding, The transcript of this gene was highly unusual as it was non-coding, and produced extremely small transcripts (22nt) from hairpin structured and produced extremely small transcripts (22nt) from hairpin structured RNA precursors.RNA precursors.

•Second microRNA, let-7 was also cloned from C. elegans (Reinhart et al, Second microRNA, let-7 was also cloned from C. elegans (Reinhart et al, 2000).2000).

•There are currently 474 human cloned and characterised microRNA There are currently 474 human cloned and characterised microRNA sequences deposited in the miRBase database sequences deposited in the miRBase database (http://microrna.sanger.ac.uk/sequences/)(http://microrna.sanger.ac.uk/sequences/)

•MicroRNAs primarily function as translational repressors by binding to MicroRNAs primarily function as translational repressors by binding to complementary target sequences in the 3’ UTR (untranslated region) of complementary target sequences in the 3’ UTR (untranslated region) of mRNA. mRNA.

Brief historyBrief history

•Between 10–30% of all human genes are a target for Between 10–30% of all human genes are a target for microRNA regulation (John et al, 2004; Lewis et al, microRNA regulation (John et al, 2004; Lewis et al, 2005). 2005).

•A single target gene often contains putative binding A single target gene often contains putative binding sites for multiple microRNAs that can bind cooperatively sites for multiple microRNAs that can bind cooperatively ,allowing microRNAs to form complex regulatory control ,allowing microRNAs to form complex regulatory control networks. networks.

•microRNAs play key regulatory roles in control of microRNAs play key regulatory roles in control of haematopoiesis, developmental timing, cell haematopoiesis, developmental timing, cell differentiation, apoptosis, cell proliferation and organ differentiation, apoptosis, cell proliferation and organ development as well as in cancer, infectious disease, development as well as in cancer, infectious disease, genetic disorders (Lin et al, 2006) and even heart genetic disorders (Lin et al, 2006) and even heart disease (van Rooij et al, 2006).disease (van Rooij et al, 2006).

The majority of human microRNAs are encoded within The majority of human microRNAs are encoded within introns of coding or non-coding mRNAs whilst others are introns of coding or non-coding mRNAs whilst others are located within the exons of non-coding mRNAs or within located within the exons of non-coding mRNAs or within the 3’UTR sequence of mRNA (Rodriguez et al, 2004).the 3’UTR sequence of mRNA (Rodriguez et al, 2004).

microRNA biosynthesis and microRNA biosynthesis and functionfunction

•MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as large MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as large polyadenylated pri-microRNAs.polyadenylated pri-microRNAs.

•RNAPII catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA Yang CGFR 16:397,

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Pri-microRNAs are cleaved within the nucleus by Drosha, Pri-microRNAs are cleaved within the nucleus by Drosha, an RNaseIII-type nuclease, to form pre-microRNA 60–70 an RNaseIII-type nuclease, to form pre-microRNA 60–70 nucleotide hairpin structures .nucleotide hairpin structures .

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• Drosha requires the cofactor DiGeorge syndrome Drosha requires the cofactor DiGeorge syndrome critical region 8 gene (DGCR8) in humans (Yeom et al, critical region 8 gene (DGCR8) in humans (Yeom et al, 2006). 2006).

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The pre-microRNAs are exported from the nucleus to the The pre-microRNAs are exported from the nucleus to the cytoplasm by Exportin5 (Zeng, 2006).cytoplasm by Exportin5 (Zeng, 2006).

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•The cytoplasmic pre-microRNA is further cleaved to form an The cytoplasmic pre-microRNA is further cleaved to form an asymmetric duplex intermediate (microRNA: microRNA*) by Dicer, asymmetric duplex intermediate (microRNA: microRNA*) by Dicer, another RNaseIII-type enzyme. Similar to Drosha, cofactors such as another RNaseIII-type enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in humans) are necessary for Dicer activity (Lee et TRBP and PACT (in humans) are necessary for Dicer activity (Lee et al, 2006). al, 2006).

microRNA:microRNA* duplex is in turn loaded into the microRNA:microRNA* duplex is in turn loaded into the miRNA-induced silencing complex miRISCmiRNA-induced silencing complex miRISC

•The consequence of miRISC-loaded microRNAs is largely dependent The consequence of miRISC-loaded microRNAs is largely dependent upon the degree of complimentarity between the microRNA and its upon the degree of complimentarity between the microRNA and its target gene.target gene.

•It leads to either degradation of mRNA or blockage of translation It leads to either degradation of mRNA or blockage of translation without degradation.without degradation.

Cell, Vol. 116, 281–297, January 23, 2004

The choice of posttranscriptional mechanisms is not determined by whether the small silencing RNA originated an siRNA or a miRNA but instead is determined by the identity of the target.

Aberrant expression of microRNAAberrant expression of microRNA

•The majority of human microRNAs are located at cancer-The majority of human microRNAs are located at cancer-associated genomic regions (Calin et al, 2004a).associated genomic regions (Calin et al, 2004a).

•microRNA expression profiling can distinguish cancers microRNA expression profiling can distinguish cancers according to diagnosis and developmental stage of the tumour according to diagnosis and developmental stage of the tumour to a greater degree of accuracy than traditional gene to a greater degree of accuracy than traditional gene expression analysis (Lu et al, 2005).expression analysis (Lu et al, 2005).

•MicroRNAs play a direct role in oncogenesis as they can MicroRNAs play a direct role in oncogenesis as they can function as both oncogenes (e.g. MIRN155 and members of function as both oncogenes (e.g. MIRN155 and members of MIRN17–92 cluster) and tumour suppressor molecules [e.g. MIRN17–92 cluster) and tumour suppressor molecules [e.g. MIRN15A (miR-15a) and MIRN16-1 (miR-16-1)]. MIRN15A (miR-15a) and MIRN16-1 (miR-16-1)].

•Aberrant expression of specific microRNAs has now been Aberrant expression of specific microRNAs has now been associated with many types of cancer including solid and associated with many types of cancer including solid and haematopoietic tumours. haematopoietic tumours.

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microRNA expression in leukaemiamicroRNA expression in leukaemia•Expression levels of MIRN15A and MIRN16-1, encoded within the Expression levels of MIRN15A and MIRN16-1, encoded within the 13q14 region, were downregulated in 75% of CLL cases that 13q14 region, were downregulated in 75% of CLL cases that harboured this chromosomal abnormality.harboured this chromosomal abnormality.

•These microRNAs were subsequently shown to target BCL2 and These microRNAs were subsequently shown to target BCL2 and to induce apoptosis in vitro, suggesting they have tumour-to induce apoptosis in vitro, suggesting they have tumour-suppressor role in CLL (Cimmino et al, 2005).suppressor role in CLL (Cimmino et al, 2005).

• MIRN16-1 negatively regulates cellular growth and cell cycle MIRN16-1 negatively regulates cellular growth and cell cycle progression (Linsley et al, 2007). progression (Linsley et al, 2007).

•A follow-up study (Calin et al, 2005) of 94 CLL cases, defined a A follow-up study (Calin et al, 2005) of 94 CLL cases, defined a prognostically significant 13-gene microRNA signature by prognostically significant 13-gene microRNA signature by expression profiling.expression profiling.

•Moreover two of the CLL patients were found to have germline Moreover two of the CLL patients were found to have germline mutations in the MIRN16-1/MIRN15A precursor sequence that mutations in the MIRN16-1/MIRN15A precursor sequence that resulted in reduced expression levels of these microRNAs both in resulted in reduced expression levels of these microRNAs both in vitro and in vivo. vitro and in vivo.

TRENDS in genetics vol22, no3 March 2006

DesignDesign

• n=94 CLL pt. samples for initial datasetn=94 CLL pt. samples for initial dataset

• Known clinical outcome data and ZAP-70 Known clinical outcome data and ZAP-70 and IgVh mutation status (retrospective)and IgVh mutation status (retrospective)– Zap-70 - >20% or < 20%Zap-70 - >20% or < 20%– IgVh status – mutated or unmutated based on IgVh status – mutated or unmutated based on

sequencing (>98% homology cutoff)sequencing (>98% homology cutoff)

• microRNA microarray analysis of 245 microRNA microarray analysis of 245 miRNAs (a subset of known miRNA)miRNAs (a subset of known miRNA)

NEJM 353:1793, 2005

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94 CLLpatients

Group 1N=36

ZAP-70 +Unmutated IgVh

Group 3N=1

ZAP-70 -Unmutated IgVh

Group 4N=47

ZAP-70 -Mutated IgVh

Group 2N=10

ZAP-70 +Mutated IgVh

miRNA micro-array(supervised)

13 miRNA signature (all mature):discriminates group 1 from group 4 (p < 0.01)

NEJM 353;17 0CT 2005

Of 13 microRNAs, 9 were significantly overexpressed in group 1, the group with a poor prognosis

NEJM 353;17 0CT 2005

Of 13 microRNAs, 9 were significantly overexpressed in group 1, the group with a poor prognosis

ValidationValidation50 CLLpatients

Group 1N=36

ZAP-70 +Unmutated IgVh

Group 4N=47

ZAP-70 -Mutated IgVh

miRNA microarray:Applied 13 miRNA signature

algorithm

50/50 correctly classified

94 CLL Patients(41 treated)

Time to Initial Treatment Data

Supervised PAM“Survival” Analysis:

Time to Initial Treatment

Short44 +/- 39 months

Long88 +/- 42 months

9 miRNA signature:discriminates pts with long vs. short interval

from Dx to treatment(ended up being a subset of 1st 13 miRNA signature)

NEJM 353;17 0CT 2005

The significance of the differences in the Kaplan–Meier curves increased if we restricted the analyses to the 83 patients in the two maingroups (groups 1 and 4) (P values decreased from <0.01 to <0.005).

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miRNA signatures identify miRNA signatures identify prognostic groups, what about prognostic groups, what about pathogenesis?pathogenesis?

Question:Question:

Are sequence alterations in genomic Are sequence alterations in genomic DNA responsible for observed DNA responsible for observed

differences in miRNA expression?differences in miRNA expression?

Genomic Alterations in Genomic Alterations in miRNAmiRNA• Sequenced 42 miRNA genes (including all Sequenced 42 miRNA genes (including all

from identified signatures)from identified signatures)• Germline or somatic mutations in 11/75 Germline or somatic mutations in 11/75

CLL samples (15%)CLL samples (15%)• All mutations located in 5/42 miRNAs (12%)All mutations located in 5/42 miRNAs (12%)

– miR-16-1miR-16-1, miR-27b, miR-29b-2, miR-187, miR-, miR-27b, miR-29b-2, miR-187, miR-206206

• 0/160 normal donors (without cancer) had 0/160 normal donors (without cancer) had these miRNA genomic changesthese miRNA genomic changes

• Of 11 pts with abnormal microRNA Of 11 pts with abnormal microRNA sequence, 8 (73%) had a known personal sequence, 8 (73%) had a known personal or 1or 1stst degree relative with cancer degree relative with cancer

NEJM 353;17 0CT 2005

microchip analysis and Northern blotting showed that CLL cells from both patients had a substantial reduction in the expression of miR-16-1 as compared with that of normal CD5+ cells

In Vitro Confirmation that mutations in In Vitro Confirmation that mutations in genomic DNA encoding miRNA affects miRNA genomic DNA encoding miRNA affects miRNA expressionexpression• Cloned genomic sequence encoding both Cloned genomic sequence encoding both

miR-16-1 and miR-15a (both wt and mutant miR-16-1 and miR-15a (both wt and mutant C->T +7)C->T +7)

• Ligated into expression vector pSR-GFP-NeoLigated into expression vector pSR-GFP-Neo• Transfected (lipofectamine) into 293 cellsTransfected (lipofectamine) into 293 cells• Assessed miRNA expression by northernAssessed miRNA expression by northern

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To identify a possible molecular effect of the C->T substitution, vectors containing either the wild-type allele of the miR-15a–miR-16-1 cluster or the mutated allele were prepared. The 293 cells, were transfected with the vectors.

•Significant association between the expression of certain Significant association between the expression of certain microRNAs and the expression of ZAP-70, the mutational status microRNAs and the expression of ZAP-70, the mutational status of of IgVH, and the time between diagnosis and initial IgVH, and the time between diagnosis and initial treatment. treatment.

•A unique 13-gene molecular signature is associated with each A unique 13-gene molecular signature is associated with each prognostic factor. prognostic factor.

•microRNA expression can be included in the markers with microRNA expression can be included in the markers with prognostic significance in CLL.prognostic significance in CLL.

•microRNA signature may be relevant to the pathogenesis of microRNA signature may be relevant to the pathogenesis of CLL.CLL.

Mechanism active in CLL pathogenesis?Mechanism active in CLL pathogenesis?miR-15a and miR-16-1 induce apoptosis miR-15a and miR-16-1 induce apoptosis by targeting bcl2by targeting bcl2

• Bcl2 protein expression is inversely correlated with Bcl2 protein expression is inversely correlated with miR-15a and miR-16-1 expression in CLL samplesmiR-15a and miR-16-1 expression in CLL samples

• Bcl2 mRNA is a direct target of miR-15a and miR-16-1 Bcl2 mRNA is a direct target of miR-15a and miR-16-1

• miR-15a or miR-16-1 induce apoptosis in bcl2+ CLLcell miR-15a or miR-16-1 induce apoptosis in bcl2+ CLLcell line, while mutant miR-16-1 c->T+7 does notline, while mutant miR-16-1 c->T+7 does not

Cimmino PNAS 102:13944, 2005

Gene therapy and RNA interferenceGene therapy and RNA interference

•Gene therapy, intends to provide therapeutic merit by Gene therapy, intends to provide therapeutic merit by introducing genetic material (DNA or RNA) encoding a introducing genetic material (DNA or RNA) encoding a protein that is missing or defective into a patient’s cells or protein that is missing or defective into a patient’s cells or tissues. tissues.

•A hallmark of gene therapy is the efficient delivery of A hallmark of gene therapy is the efficient delivery of these nucleic acids via the use of shuttle vectorsthese nucleic acids via the use of shuttle vectors

•Either nonviral, such as liposomes or nanoparticles, or Either nonviral, such as liposomes or nanoparticles, or derived from genetically modified viruses. ( adeno-derived from genetically modified viruses. ( adeno-associated virus (AAV), adenovirus (Ad) and lentivirus)associated virus (AAV), adenovirus (Ad) and lentivirus)

RNAi gene therapy applicationRNAi gene therapy application

•Viral infections:Viral infections:

- HIV- HIV

- Hep B- Hep B

- Hep C- Hep C

- RSV- RSV

• CancerCancer

• Neurodegenerative disorders:Neurodegenerative disorders:

- Spinocerebellar Ataxias- Spinocerebellar Ataxias

- Huntington disease- Huntington disease

- alzheimer disease- alzheimer disease

• Ocular disorders (Macular degeneration)Ocular disorders (Macular degeneration)

• Stem cell biology and therapy Stem cell biology and therapy

ASH education book 2007

SummarySummary• miRNA may act as tumor suppressors or miRNA may act as tumor suppressors or

oncogenesoncogenes• miRNA profiles distinguish human tumor typesmiRNA profiles distinguish human tumor types• miRNA signature correlates with ZAP-70/Ig Vh miRNA signature correlates with ZAP-70/Ig Vh

mutation status prognosis and length of time to mutation status prognosis and length of time to initial treatment in CLLinitial treatment in CLL

• Alterations in genomic DNA encoding miRNA exist Alterations in genomic DNA encoding miRNA exist in CLL pts, and in 16-1 causes decreased miRNAin CLL pts, and in 16-1 causes decreased miRNA

• miR-15a and miR-16-1 directly target bcl2, and miR-15a and miR-16-1 directly target bcl2, and their absence may contribute to the pathogenesis their absence may contribute to the pathogenesis of CLLof CLL

• miRNA or miRNA-like sequences may be used miRNA or miRNA-like sequences may be used therapeutically to target bcl2 or other oncogenes therapeutically to target bcl2 or other oncogenes in the futurein the future

Cimmino PNAS 102:13944, 2005

Indications for Treatment based on Indications for Treatment based on Current Knowledge – IWCLL Current Knowledge – IWCLL Guidelines (1996)Guidelines (1996)• Advanced stageAdvanced stage

– Improved survival with therapy restricted to Rai Improved survival with therapy restricted to Rai III/IV stage or rapid progression of diseaseIII/IV stage or rapid progression of disease

• SymptomsSymptoms– Decreased PSDecreased PS– Debilitating constitutional SxDebilitating constitutional Sx– Complications/Sx from spleen, liver, LN Complications/Sx from spleen, liver, LN

enlargementenlargement

• Disease activityDisease activity– Lymphocyte doubling time < 6 monthsLymphocyte doubling time < 6 months– Cytopenias (BM involvement or AI)Cytopenias (BM involvement or AI)– Rapid LN enlargementRapid LN enlargement

Binet et al. Blood DOI 10.1182, 2005