MicroRNA’s applications in medicine

2. MicroRNA’s applications in medicine

3. microRNA Related Publications Source: PubMed

4. lin-14; lin-4 relationship 1.lin-4 encodes small RNAs with antisense complementarity to lin-14 3’ UTR.[Cell, 75, 843 (1993)] 2. The LIN-14 protein concentration drops in late development, whereas the lin-4 RNA increases. 3. MECHANISM: lin-4 RNA increases in later development and inhibits translation of the lin-14 gene and thus specifies the pattern of late-stage development. lin-14 mRNA translation lin-4 RNA

5. lin-4 RNA Structure • lin-4 encodes two mRNAs: 61 and 22. • 61 forms a stem-loop structure and is the precursor of 22 nt • The red nt sequence is the 22 nt microRNA. Cell, 106, 23 (2001)

6. microRNA (miRNA) Cell, 116, 281, 2004

8. miRNA biogenesis pathway

9. following debranching of lariat-structures known as mirtrons, (iii) • alternative folding of transfer RNAs (tRNAs) or small nucleolar RNAs (snoRNAs), or (iv) • By tRNAse Z cleavage of pri-miRNAs containing tRNA-like structures linked to pre-miRNAstem-loops Annu. Rev. Microbiol. 2010. 64:123–41

10. Formation of MicroRNA cont. RNA Pol II—intron encoded miRNA RNA Pol III—independently encoded miRNA minor group

11. miRNA Gene • multiple isoforms • 12 loci for let‑7‑family miRNAs • Approximately 50% of mammalian miRNA loci are found in close proximity • a single polycistronic transcription unit

12. miRNA Gene

13. Evolutionarily conserved small noncoding RNAs in eukaryotes phylogenetically conserved; ~55%

14. MIRNA feature • ~30% of mammalian genes are regulated by miRNAs • miRNAs are often expressed at more than 2,000 copies per cell in a cell-type and tissue-specific manner NATURE REVIEWS | MICROBIOLOGY-VOLUME 4 -2006 | 651

15. miRNA function Variation • Repressing Translation • with individual sites less than a half and often by less than a third • many more targets dramatically repressed • modest mRNA destabilization (2-fold) • heterochromatin formation • Enhancing translation • HCV • Enhancing gene expression aRNAs (small activating RNAs) • Promoter targeting

16. miRNA Function Mode of Action of miRNAs in Plants and Animals

17. miRNA Target Sites nucleotides 2–8 prearranged in a geometry resembling an A-form helix

18. Processing bodies are sites of storage and/or degradation of mRNA

19. microRNAs and Pbodies • Localization of RISC-bound mRNAs often to cytoplasmic processing bodies (Pbodies) • which exclude the translational machinery • mRNA remodeling • decapping • de-adenylation, as well as exonucleases

20. Regulation of miRNA • Alternative cleavage and polyadenylation • In proliferating cells the shorter UTRs • only of conserved miRNA sites • longer isoforms that dominate in nonproliferating cells • Blocking by the binding of Deadend RNA-binding protein in the germline • Site masking by protein • blockage of Drosha processing • interference with Dicer processing • controlled at the nuclear export step • uridylation of pre-miRNA (14 u in 3’ end)

21. Target-Prediction Tools

22. Target-Prediction Tools

23. microRNA; small is mighty!

24. MicroRNAs in disease diagnostics • miRNAsdisplayed high stability in paraffin-embedded tissues from clinical samples or in human plasma • miRNA profiles could distinguish a tumor’s developmental origin • miRNA signatures were further used to define subtypes of cancers • such as the distinction between basal and luminal breast cancers • miRNA expression profiles to distinguish forms of heart disease • muscular disorders • neurodegenerative diseases

25. Rosetta Genomics diagnostic panels

26. MicroRNAs have been reported to be: • Differentially expressed in tissues • Critical in the development of organisms • Associated with oncogenes • Involved in viral infection processes • Neurodegenerative diseases • Cardiovascular diseases

27. Differential expression of MicroRNAs • MicroRNAs (miRNAs) are an abundant class of negative gene regulators that have been shown to control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis. • Different tissues express different types of miRNAs It indicates that: • Specific miRNAs have specific roles in different conditions

28. MicroRNAs regulate cell cycle

29. MicroRNAs regulate ES

30. MicroRNAs regulate Hematopoisis

31. MicroRNAs regulate Myogenesis

32. MicroRNAs regulate Neurogenesis

33. MicroRNAs regulate osteogenesis & Skin

34. Distribution of US patents in miRNA’s fields

35. MicroRNAs in Clinic

36. Some MicroRNAs Pepline

37. STEM CELLS technology research center projects

39. miRNA expression on metastatic breast cancer

40. Breast cancer • The most incident malignancy in women • The leading cause of cancer-related death in women in developed countries • Accounts for one-sixth of cancer deaths in the United States. • 1.15 million new breast cancer cases and over 500,000 deaths reported around the world annually In Iran: • The incidence rate is 7000 case annually • The incidence age is 10 years lower in Iran(35-45y) • 60% of all patients treated for early-stage disease ultimately developed recurrence, mostly metastatic (30% in the world) • 15.8% Triple-Negative Breast cancer(TNBC) • Survival time in 65-70% of cases is <=5 years

41. Selection of Wnt signaling pathway interfering in metastasis of breast cancer Oncotarget 2011.1: 563-576

42. CTNNB1(β-catenin) RhoA ROCK1 • Signaling pathway study(selection of target genes) 2. miRNA prediction • miRWalk, TargetScan 6.2 ، Miranda ، PicTar,MicroCosm • miRWalk , miR Path miR-340 miR-381

43. Example of Bioinformatics: Target Scan microRNA.org Pictar

44. Results of Luciferase assay Genes expression in MCF-7 and MDA-MB-231 • miRs expression in MCF-7 and MDA-MB-231

45. Compare of Gene expression before and after miRNA transduction • Migration Assay Invasion assay

46. Pathological interpretation • 10 nude female mice • 5mice in each group • Injection of 5×105 cells • MDA-miR-340/ control vector • To the tail vain • 28 days follow up • Scarify the mice • Separation of Lung Liver, Bone and Brain Liver: Diffused and patchy hepatic necrosis Presenting of inflammatory cells Geographical liver infarction Lung: filling of alveolar space by diffuse hemorrhage and collapse Acute and severe inflammation Brain: Negative Bone: Negative

47. 1 Suggestions for future • Usage of bioinformatics for miRNA predictions • Confirmation of prediction • Stem-loop assumption for miRNA expression analysis • New therapeutics approaches base on molecular techniques for metastatic Breast cancer • Tumor suppressor miRNAs for adjuvant therapy • New and less harmful miRNA delivery systems • Directly ushering miRNAs to the Cancerous cells

48. microRNA-mediated suppression of Androgen receptor pathway in prostate cancer

49. Prostate Cancer • Prostate cancer (PC) is one of the major problems in public health around the world. • It is estimated that 1 in every 6 men may develop PC and 1 in 30 will die of this metastatic disease. • About 80% of all men in their eighties show pathologic evidence of prostate cancer when they died

50. Cancer death rate among men 1930-2006