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microRNAs

microRNAs. Small molecules, big functions Ali Bazargan. Brief history. The first described microRNA, lin-4 was cloned and characterised as a translational repressor of developmental timing from Caenorhabditis. elegans by Lee et al (1993) and Wightman et al (1993).

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microRNAs

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  1. microRNAs Small molecules, big functions Ali Bazargan

  2. Brief history The first described microRNA, lin-4 was cloned and characterised as a translational repressor of developmental timing from Caenorhabditis. elegans by Lee et al (1993) and Wightman et al (1993). The transcript of this gene was highly unusual as it was non-coding, and produced extremely small transcripts (22nt) from hairpin structured RNA precursors. Second microRNA, let-7 was also cloned from C. elegans (Reinhart et al, 2000). There are currently 474 human cloned and characterised microRNA sequences deposited in the miRBase database (http://microrna.sanger.ac.uk/sequences/) MicroRNAs primarily function as translational repressors by binding to complementary target sequences in the 3’ UTR (untranslated region) of mRNA.

  3. Brief history Between 10–30% of all human genes are a target for microRNA regulation (John et al, 2004; Lewis et al, 2005). A single target gene often contains putative binding sites for multiple microRNAs that can bind cooperatively ,allowing microRNAs to form complex regulatory control networks. microRNAs play key regulatory roles in control of haematopoiesis, developmental timing, cell differentiation, apoptosis, cell proliferation and organ development as well as in cancer, infectious disease, genetic disorders (Lin et al, 2006) and even heart disease (van Rooij et al, 2006).

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

  5. microRNA biosynthesis and function

  6. MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as large polyadenylated pri-microRNAs. • RNAPII catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA Yang CGFR 16:397, 2005

  7. BJH 137, 503-512 2007 Pri-microRNAs are cleaved within the nucleus by Drosha, an RNaseIII-type nuclease, to form pre-microRNA 60–70 nucleotide hairpin structures .

  8. BJH 137, 503-512 2007 • Drosha requires the cofactor DiGeorge syndrome critical region 8 gene (DGCR8) in humans (Yeom et al, 2006).

  9. BJH 137, 503-512 2007 The pre-microRNAs are exported from the nucleus to the cytoplasm by Exportin5 (Zeng, 2006).

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

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

  12. The consequence of miRISC-loaded microRNAs is largely dependent upon the degree of complimentarity between the microRNA and its target gene. • It leads to either degradation of mRNA or blockage of translation without degradation.

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

  14. Aberrant expression of microRNA The majority of human microRNAs are located at cancer-associated genomic regions (Calin et al, 2004a). microRNA expression profiling can distinguish cancers according to diagnosis and developmental stage of the tumour to a greater degree of accuracy than traditional gene expression analysis (Lu et al, 2005). MicroRNAs play a direct role in oncogenesis as they can function as both oncogenes (e.g. MIRN155 and members of MIRN17–92 cluster) and tumour suppressor molecules [e.g. MIRN15A (miR-15a) and MIRN16-1 (miR-16-1)]. Aberrant expression of specific microRNAs has now been associated with many types of cancer including solid and haematopoietic tumours.

  15. BJH 137, 503-512 2007

  16. microRNA expression in leukaemia Expression levels of MIRN15A and MIRN16-1, encoded within the 13q14 region, were downregulated in 75% of CLL cases that harboured this chromosomal abnormality. These microRNAs were subsequently shown to target BCL2 and to induce apoptosis in vitro, suggesting they have tumour-suppressor role in CLL (Cimmino et al, 2005). MIRN16-1 negatively regulates cellular growth and cell cycle progression (Linsley et al, 2007). A follow-up study (Calin et al, 2005) of 94 CLL cases, defined a prognostically significant 13-gene microRNA signature by expression profiling. Moreover two of the CLL patients were found to have germline mutations in the MIRN16-1/MIRN15A precursor sequence that resulted in reduced expression levels of these microRNAs both in vitro and in vivo.

  17. TRENDS in genetics vol22, no3 March 2006

  18. Design • n=94 CLL pt. samples for initial dataset • Known clinical outcome data and ZAP-70 and IgVh mutation status (retrospective) • Zap-70 - >20% or < 20% • IgVh status – mutated or unmutated based on sequencing (>98% homology cutoff) • microRNA microarray analysis of 245 miRNAs (a subset of known miRNA) NEJM 353:1793, 2005

  19. NEJM 353;17 0CT 2005

  20. miRNA micro-array (supervised) 13 miRNA signature (all mature): discriminates group 1 from group 4 (p < 0.01)

  21. Of 13 microRNAs, 9 were significantly overexpressed in group 1, the group with a poor prognosis NEJM 353;17 0CT 2005

  22. Of 13 microRNAs, 9 were significantly overexpressed in group 1, the group with a poor prognosis NEJM 353;17 0CT 2005

  23. Validation 50/50 correctly classified

  24. 9 miRNA signature: discriminates pts with long vs. short interval from Dx to treatment (ended up being a subset of 1st 13 miRNA signature)

  25. The significance of the differences in the Kaplan–Meier curves increased if we restricted the analyses to the 83 patients in the two main groups (groups 1 and 4) (P values decreased from <0.01 to <0.005). NEJM 353;17 0CT 2005

  26. NEJM 353;17 0CT 2005

  27. miRNA signatures identify prognostic groups, what about pathogenesis? Question: Are sequence alterations in genomic DNA responsible for observed differences in miRNA expression?

  28. Genomic Alterations in miRNA • Sequenced 42 miRNA genes (including all from identified signatures) • Germline or somatic mutations in 11/75 CLL samples (15%) • All mutations located in 5/42 miRNAs (12%) • miR-16-1, miR-27b, miR-29b-2, miR-187, miR-206 • 0/160 normal donors (without cancer) had these miRNA genomic changes • Of 11 pts with abnormal microRNA sequence, 8 (73%) had a known personal or 1st degree relative with cancer

  29. NEJM 353;17 0CT 2005

  30. 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

  31. In Vitro Confirmation that mutations in genomic DNA encoding miRNA affects miRNA expression • Cloned genomic sequence encoding both miR-16-1 and miR-15a (both wt and mutant C->T +7) • Ligated into expression vector pSR-GFP-Neo • Transfected (lipofectamine) into 293 cells • Assessed miRNA expression by northern NEJM 353;17 0CT 2005

  32. 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. NEJM 353;17 0CT 2005

  33. Significant association between the expression of certain microRNAs and the expression of ZAP-70, the mutational status of IgVH, and the time between diagnosis and initial treatment. A unique 13-gene molecular signature is associated with each prognostic factor. microRNA expression can be included in the markers with prognostic significance in CLL. microRNA signature may be relevant to the pathogenesis of CLL.

  34. Mechanism active in CLL pathogenesis?miR-15a and miR-16-1 induce apoptosis by targeting bcl2 • Bcl2 protein expression is inversely correlated with miR-15a and miR-16-1 expression in CLL samples • Bcl2 mRNA is a direct target of miR-15a and miR-16-1 • miR-15a or miR-16-1 induce apoptosis in bcl2+ CLLcell line, while mutant miR-16-1 c->T+7 does not Cimmino PNAS 102:13944, 2005

  35. Gene therapy and RNA interference Gene therapy, intends to provide therapeutic merit by introducing genetic material (DNA or RNA) encoding a protein that is missing or defective into a patient’s cells or tissues. A hallmark of gene therapy is the efficient delivery of these nucleic acids via the use of shuttle vectors Either nonviral, such as liposomes or nanoparticles, or derived from genetically modified viruses. ( adeno-associated virus (AAV), adenovirus (Ad) and lentivirus)

  36. RNAi gene therapy application Viral infections: - HIV - Hep B - Hep C - RSV Cancer Neurodegenerative disorders: - Spinocerebellar Ataxias - Huntington disease - alzheimer disease Ocular disorders (Macular degeneration) Stem cell biology and therapy

  37. ASH education book 2007

  38. Summary • miRNA may act as tumor suppressors or oncogenes • miRNA profiles distinguish human tumor types • miRNA signature correlates with ZAP-70/Ig Vh mutation status prognosis and length of time to initial treatment in CLL • Alterations in genomic DNA encoding miRNA exist in CLL pts, and in 16-1 causes decreased miRNA • miR-15a and miR-16-1 directly target bcl2, and their absence may contribute to the pathogenesis of CLL • miRNA or miRNA-like sequences may be used therapeutically to target bcl2 or other oncogenes in the future

  39. Cimmino PNAS 102:13944, 2005

  40. Indications for Treatment based on Current Knowledge – IWCLL Guidelines (1996) Advanced stage Improved survival with therapy restricted to Rai III/IV stage or rapid progression of disease Symptoms Decreased PS Debilitating constitutional Sx Complications/Sx from spleen, liver, LN enlargement Disease activity Lymphocyte doubling time < 6 months Cytopenias (BM involvement or AI) Rapid LN enlargement Binet et al. Blood DOI 10.1182, 2005

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