1. GOOD MORNING

2. miRNA and Its Applications BY GROUP 15 3. GROUP MEMBERS NAVYA RAJEEV KARTIK CHADAR VIBHUTI VAIBHAV NAYAN GUPTA ROHIT SHRIVASTAVA 4. INTRODUCTION Small noncoding RNA molecule(~22 nucleotides). Found in plants, animals, virus. They are regulatory RNAs combinatorial regulation is the key feature. Human genome may encode over 1000 miRNAs. 5. MICRO RNA HISTORY AND DISCOVERY 6. Discovery of the first miRNA: lin-4 Lin-4 was the first miRNA to be discovered [1993] By the joint efforts of VictorAmbros on lin-4 [1987] and Gary Ruvkun on lin-14 [1988] Study of the gene lin-14 in Caenorhabditis elegans development Mutation in this gene causes failure in temporal devlopment missing some adult structures,and are unable of laying eggs Fergunsson et al., at Hortitzs lab, found that a suppressor mutation in the gene lin-14 was able to revert the null-lin-4 mutation phenotype null mutations in lin-14 gene caused an exactly opposite phenotype of the null-lin-4 mutations indicated that lin-4 could negatively regulate lin-14 Doesnt code protein because lack start and stop codon In December 1993, in the same issue of Cell,Ambros and Ruvkun independently reported that the small and non-protein coding transcript lin4 regulates lin-14. 7. VICTOR AMBROS AND HIS GROUP Rhonda Feinbaum 8. Discovery of a second microRNA: Let-7 Lin-4 was the first miRNA to be discovered [2000] let-7 was a 21 nt RNA controlling L4-to-adult transition of larval development in same C. elegans Loss of let-7 activity causes reappearance of larval cell fates during adult stage of development, while increased let-7 activity causes precocious expression of adult fates let-7 RNA was detected in vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from plant and unicellular organisms Let-7 family within humans comprises 12 miRNAs Presently, thousands of miRNAs had been identified in humans and other species 9. Bruce Wightman GARY RUVKUN AND HIS GROUP 10. Biogenesis of microRNA BY,KARTIK CHADAAR ROLL NO. 43 11. Steps in biogenesis Transcription Nuclear Processing Nuclear Export Cytoplasmic Processing AND there, it is finally ready!!!! 12. Gene for miRNA Majority of the miRNA genes are intergenic or oriented antisense to neighboring genes and are therefore suspected to be transcribed as independent units. However, in some cases a microRNA gene is transcribed together with its host gene; this provides a means for coupled regulation of miRNA and protein-coding gene. 40% of miRNA genes may lie in the introns of protein and non-protein coding genes or even in exons of long nonprotein-coding transcripts. 13. TRANSCRIPTION transcribed by RNA polymerase II (Pol II) resulting transcript is capped with a specially modified nucleotide at the 5 end i.e. 7Mguanosine, polyadenylated with a poly(A) tail. This forms the first miRNA precursor called primary-miRNA or pri-miRNA. 14. Primary miRNA HAIR PIN loop structure: Double Stranded RNA structure. Contains around 70 nucleotides. Two hairpin loops flanked by regions required for efficient processing. Acted upon by Drosha and Pasha. 15. NUCLEAR Processing The pri-miRNA transcript is then cleaved by the endonuclease III enzyme Drosha. Drosha is accompanied by another protein named DiGeorge Syndrome Critical Region 8(DGCR8), or to be simple in terms Pasha. Drosha and Pasha form a complex which is named Microprocessor complex. 16. In this complex, DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base. The product resulting has a two-nucleotide overhang at its 3 end; it has 3' hydroxyl and 5' phosphate groups. It is often termed as a pre-miRNA (precursor-miRNA). 17. Pre-miRNA Consists of: 3hydroxl and 5phosphate group. 2 nucleotide overhung at 3 end. 18. ALSO Pre-miRNAs that are spliced directly out of introns, bypassing the Microprocessor complex, are known as "Mirtrons." Originally thought to exist only in Drosophila and C. elegans, mirtrons have now been found in mammals 19. Nuclear Export Pre-miRNA hairpins are exported out of the nucleus in a process involving the nucleocytoplasmic shuttler Exportin-5. Exportin-5-mediated transport to the cytoplasm is energy- dependent, using GTP bound to the Ran protein. 20. Cytoplasmic Processing In the cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer. This endoribonuclease interacts with the 3' end of the hairpin and cuts [the pre-miRNA approximately 19bp from the Drosha cut site (AMbion)] away the loop joining the 3' and 5' arms, yielding an imperfect miRNA:miRNA duplex about 22 nucleotides in length. 21. Although either strand of the duplex may potentially act as a functional miRNA, only one strand is usually incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact. RISC is also known as a microRNA ribonucleoprotein complex (miRNP) 22. Generally, only one strand is incorporated into the miRISC, selected on the basis of its thermodynamic instability and weaker base-pairing relative to the other strand. damiRNA Duplex Guide Strand: Thermodynamically more stable Passenger Strand: Less Thermodynamic Stability Forms functional miRNA Mostly Degraded 23. RNA SILENCING The mature miRNA is part of an active RNA-induced silencing complex (RISC) containing Dicer and many associated proteins. Members of the Argonaute (Ago) protein family are central to RISC function. They bind the mature miRNA and orient it for interaction with a target mRNA 24. Gene silencing in plants and animals mRN A Ribosom e 25. Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. Unlike plant microRNAs, the animal microRNAs target a diverse set of genes 26. On the lighter side 27. Interaction of microRNA with protein translation process. Several (from nine documented) mechanisms of translation repression are shown: M1) on the initiation process, preventing assembling of the initiation complex or recruiting the 40S ribosomal subunit; M2) on the ribosome assembly; M3) on the translation process; M7, M8) on the degradation of mRNA 28. miRNAs occasionally also cause histone modification and DNA methylation of promoter sites, which affects the expression of target genes 29. w 30. MICRORNAs as BIOMARKERS 31. miR-20 chronic lymphocytic leukemia. miR-855-5p liver pathologies. miR-21 prognosis of pancreatic cancer. miR-326 multiple sclerosis. miR-28-3p type 2 diabetes. miR-126 angiogenesis. 32. MICRORNAs AND CANCER BY NAYAN GUPTA 33. CANCER STEM CELLS Cancer stem cells(CSCs) have been reported in many human tumors and are proposed to drive tumor initiation and progression. CSCs hare a variety of biological properties with normal somatic stem cells such as the capacity for self- renewal, the propagation of differentiated progeny, and the expression of specific cell surface markers and stem cell genes. 34. However, CSCs are different from normal stem cells in their chemoresistance and tumorigenic and metastatic activities. 35. CSCs and miRNAs Aberrant miRNA expression is associated with many human diseases including cancer. miRNAs have been implicated in the regulation of CSC properties. In the present review, we summarize the major findings on the regulation of CSCs by miRNAs and discuss recent advances that have improved our understanding of the regulation CSCs by miRNA networks and may lead to the development of miRNA therapeutics specifically targeting CSCs. 36. miRs interact with 3-UTR of mRNAs Low miR-mRNA base specificity (6-8) Each miR can potentially interact with several hundred mRNAs Function: block gene expression Pre-miRNA MicroRNA REGULATION OF GENE EXPRESSION IN CANCER CELLS/TUMORS 20-22 nt (single chain) miR-27a miR-27a RNA miR 37. COMPLEXITIES OF MiR-mRNA INTERACTIONS MULTIPLE MiRs REGULATE A SINGLE mRNA* the p21 3UTR can potentially be targeted by 266 miRs (p21 tumor suppressor) 266-miRs luc Transfected 3UTR p21 HEK293 cells 28 miRs interacted with 3-UTR; decreased luciferase activity overexpression of miRs decreased p21 protein and mRNA levels 38. INDIVIDUAL MiRs ASSOCIATED WITH MULTIPLE TUMORS miR TS/OG Tumors Let-7 Family MiR-159/16-1 cluster MiR-17-92 cluster MiR-26a MiR-34a/b/c MiR-21 TS TS OG TS/OG TS OG 10 7 7 4 6 10 TS = tumor suppressor; OG = oncogene 39. miRNA and REGULATION in CANCER 40. Chromosomal regions coding for oncogenic miRNAs that are involved in the negative regulation of a tumor suppressor gene can be amplified in association with cancer development. This amplification would result in the upregulation of oncogenic miR- NAs and silencing of tumor suppressor genes. 41. On the other hand, miRNAs targeting oncogenes are often located in fragile site, where deletions or mutations can occur, leading to the reduction or loss of miRNAs and the overexpression of their target oncogenes. 42. miRNAs are involved in tumor initiation through the regulation of CSC properties such as self-renewal ability, tumorigenicity and drug-resistance. Dysregulation of miRNA expression affects pro- cesses associated with cancer progression such as the induction of anti-apoptotic activity, drug resistance, tissue invasion, and metastasis. 43. MICRORNAs IN CANCER DIAGNOSIS 44. LEUKEMIA CANCER STEM CELLS 45. The miR-17-92 cluster functions as an oncogenic miRNA by enhancing the for- mation of Myc-driven B-cell lymphomas in a mouse model. 46. Han et al. reported that miR-29a regulates early hematopoiesis and induces AML by converting myeloid progen- itors into self-renewing leukemia stem cells via targeting several tumor suppressors and cell cycle regulators 47. miR-22 induced inhibition of some translocation gene 2 (TET2) tumor suppressor increased the methylation of TET2 target genes, such as Aim2, Hal, Igbt2, and Sp140, and resulted in positive effects on hematopoietic stem cell self- renewal and transformation. This has led to the suggestion that mir-22 is associated with myelodysplastic syndrome and hematological malignancies. 48. PROSTATE CANCER STEM CELLS 49. miR-34a is downregulated in CD44+ PCa cells purified from xenografts anD primary tumors, and that miR-34a directly regulates the expression of CD44 at the post- transcriptional level by binding to its 3UTR. Expression of miR-34a in CD44+ PCa cells inhibits tumor migration and metastasis in a xenograft model, and miR-34a inhibits Notch and Arsignaling in Pca cells, suggesting that miR-34a suppresses the self- renewal activity of CSCs in Pca cells. 50. Another miRNA that regulates CSCs properties is miR-320, which acts by directly targeting -cateninin Pca cells. miR-320 and -catenin expression is inversely correlated in CD44+ PCa cells. Furthermore, gene expression profiling of miR-320 overexpressing Pca cells showed a significant decrease in downstream target genes of the Wnt/-catenin pathway and CSC markers. 51. CONCLUSION Several studies reviewed here have shown that mirnas can function as tumor suppressors or oncogenes and play important roles in various aspects of cscproperties. In this regard, mirna sare considered to be functional markers of cscs. Therefore, a more detailed understanding of the function of mirnas in CSC biology may improve cancer treatments and possibly lead to the clinical application of mirnas in cancer diagnosis, treatment, and prognosis. 52. miRNA and DIABETES BY ROHIT SHRIVASTAVA 53. introduction Diabetes Mellitus is a complex multisystem disease that represents the most common metabolic disorder. Two Types TYPE 1 TYPE 2 54. Type I is caused by autoimmune destruction of -cell leading to insulin deficiency. In the early stages, pancreatic islets are infiltrated by immune cells, hence -cells are exposed to proinflammatory cytokines, resulting in altered insulin content, insulin secretion, and sensitisation to apoptosis. In Type II diabetes, there is problem with the insulin dependent GLUT receptors and also there is insulin resistance. 55. miRNA, Insulin Secretion And -cell function There are many miRNAs present in the B cell that regulate the activity of many genes that codes either for insulin or for the factors that regulate the insulin gene. So, any mutation or aberration in the gene of miRNA may lead to diabetes. 56. miRNA 375 miRNA- 375 is the most abundant miRNA in the islet cells It is one of the most studied miRNA. It actually negatively regulates the GSIS i.e., Glucose Stimulated Insulin Secretion from the beta-cells. 57. What is the relation of miRNA-375 to diabetes? 58. miRNA and Diabetes Inhibition of miRNA leads to the increased insulin secretion. Overexpression of miRNA leads to the decreased secretion of insulin Reason behind this is that it actually impairs the insulin signaling pathway by inhibiting myotropin (Mtpn). miRNA-375 also targets the insulin gene expression. It also downregulates the expression of phosphoinositide dependent protein kinase 1, a key component of the PIP3. It downregulates PDK 1. 59. There are many other miRNAs that regulate secretion from cells. Lets see some of them 60. miR-9 This miRNA also has inhibitory role in insuln secretion. It inhibits the transcription factor Onecut-2 which regulates Granuphilin. 61. miR-96 It decreases the expression of nucleolar complex protein 2 (Noc2), a Rab GTPase effector required for insulin exocytosis. It also upregulates granuphilin . 62. miR-124a miR-124a was earlier thought to be vital for pancreatic -cell development. It also modulates several components of the exocytotic system by directly targeting Forkhead Box Protein A2 (Foxa2)a transcription factor involved in glucose metabolism and insulin secretion. Modulation of miR-124a in MIN6 (mouse insulinoma) cells causes changes in Foxa2 and its downstream target gene PDX-1 (which regulates insulin transcription). Overexpression of mir-124a in MIN6 cells leads to increased insulin secretion in response to basal glucose concentrations and reduced secretion in response to stimulatory glucose concentrations. 63. Some other miRNAs MIN6 cells treated with proinflammatory cytokines show significant induction of miR-21, miR- 34a, and miR-146. Subsequent blockade of these miRs prevented cytokine induced reduction in GSIS and protected -cells from cytokine-induced cell death because of a reduction in the expression of the antiapoptotic protein Bcl2 and of VAMP2 (vesicle-associated membrane protein 2), which is involved in -cell exocytosis Experimental chronic exposure to the free fatty-acid palmitate mimics the adverse environmental conditions that promote failure of -cells, arising in defective GSIS. A further study found that exposure of insulin-secreting cell lines or pancreatic islets to palmitate led to an increase in miR-34a and miR-146 expression. 64. miRNAs and Hyperglycemia In diabetes, always there is a condition of Hyperglycemia . This hyperglycemia influences a large number of miRNAs and increases their expression. For example, upregulation of miR-30d Overexpression of miR-30d increases insulin gene expression. Its inhibition attenuates glucose stimulated insulin gene transcription. miR-15a promotes insulin biosynthesis by inhibiting endogenous UCP-2 (uncoupling protein-2) expression in mouse -cells. miR-335 was upregulated in the pancreatic islets of GK rats, and was shown to target the messenger RNA (mRNA) for the exocytotic protein Stxbp1. 65. miRNA in Insulin Target Cells 66. miRNAs in Liver In liver, the most abundant miRNA is miR-122. Inhibition of miR-122 in mice results in decreased hepatic fatty acid and cholesterol synthesis, along with a reduction in plasma cholesterol. miR-33a and miR-33b have been shown to regulate cholesterol homeostasis through interaction with sterol regulatory element- binding proteins. 67. MAPK/EMK Pathway 68. miRNA and Cardiac diseases 69. miR-133 miR-133 is believed to be expressed specifically in cardiac and skeletal muscle, with its function in skeletal muscle being to modulate myoblast proliferation and differentiation. Moreover, miR-133 controls cardiac hypertrophy and is downregulated in failing and hypertrophic hearts. The GLUT4 glucose transporter is the major mechanism by which glucose uptake into cardiomyocytes can be increased. Horie et al. found that miR-133 overexpression lowered GLUT4 levels and reduced insulin-induced glucose uptake in cardiomyocytes. Additionally, increased miR-133 also reduces the Krppel-like transcription factor KLF15, which induces GLUT4 expression. 70. A prolonged QT interval, an adverse cardiac feature of diabetes, can result in arrhythmias and has been suggested as an independent predictor of mortality in DM. Zhang et al. confirmed a 20% prolongation of the QT interval in diabetic rabbits compared with controls. This occurs as a result of dysfunction of multiple ion currents/channels, predominantly the I /HERG (human ether-a-go-go) channel. The same group found that levels of miR-133 and miR-1 were significantly upregulated in the hearts of diabetic rabbits compared with controls. Furthermore, miR-133 overexpression reduced HERG protein levels, while miR-133 inhibition partially reversed this. This suggests a role for miR133 dysfunction in prolonging the QT interval, and causing the resultant arrhythmias, in diabetic hearts. These studies suggest that miR-133 has two potential roles in the diabetic heart, depending upon whether expression is increased or decreased 71. miRNA and Angiogenesis 72. THERAPEUTIC POTENTIAL OF miRNA NAVYA RAJEEV 73. Only 10 years ago first human miRNA was discovered and yet miRNA based therapeutic already entered phase 2 clinical trial. 74. THERAPEUTIC miRNA MODALITIES miRNA antagonists miRNA mimics Two approaches 75. miRNA antagonists Inhibit endogen ous miRNA that show a gain of function in diseased tissue. New miRNA duplex unable to be processed by RISC The miRNA duplex is degraded Bind to active miRNA with high affinity (Binding is irreversible) Introduce highly chemically modified miRNA (anti miRNA) 76. miRNA mimics Used to restore a loss of function. Also called miRNA replaceme nt therapy Reintroduce miRNA into diseased cells that are normally present in normal cells. Reactivation of pathways for cellular welfare Block that drive the disease. 77. What makes it different from other gene therapy method..?? Face less of delivery hurdle compared with protein encoding plasmid DNA used earlier. Is highly specific Tolerated in normal tissues - as they carry the same sequence in naturally occuring equivalent and target the same gene. 78. miRNA IN THERAPEUTIC DEVELOPMENT miR 122 Hepatitis C virus miR 208 chronic heart failure miRNA antagonists miR 34 cancer let 7 - cancer miRNA mimics 79. Most clinically advanced till date targets Hepatitis C virus. miR-122 antagonist, SPC3649 is administered to hepatocytes to block replication of the virus. miR-122, binds to two closely spaced target sites within the HCV genome that is necessary to maintain the abundance of viral RNA. Induced a long-lasting suppression of viral RNA in serum. 80. A new mechanism of action, the ability to function as master regulators of the genome and an apparent lack of adverse events in normal tissue make drug target miRNA a promising technology for current and future product development. 81. THANK YOU