The Primary Study On The Injury Of Pancreas Induced By Overexpression Of MiR-375

MicroRNAs are 22nt short no-code RNA. It is derived from pre-miRNA by Dicer enzyme. Several hundreds of miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis and immune responses. MiRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts, regulating the expression of 30% gene in human. With new miRNAs were found, increasing number of people pay attention to the study on the miRNAs. Therefore, the research for miRNAs has become one of the hosttest fields in life science.There are lots of papers about function of miRNAs. But they study the function of miRNAs in the cells. There is scarcely paper about the function of miRNAs in whole body. It is inadequate that we study on the function of miRNAs only in the cell, because miRNAs act as function in the whole body. In this study we microinject the transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene into the pronucleus of fertilized oocytes to construct transgenic mice. In order to study on the injure of pancreas induced by the overexpression of miR-375.Our study includes four parts, the main methods and results are as follows: PartⅠCloning of the miR-375 and primary study on the function of the miR-375We construct the plasmid of pAAV-MCS-miR-375 (mouse miR-375). The HeLa cell line was transfected with the plasmid of pAAV-MCS-miR-375. After 48 hours the HeLa cells transfected, Total RNA was extracted from the HeLa cells using TRIzol (Invitrogen). RT-PCR analysis showed the expression of miR-375 in the transfected Hela cells. We transfected the Nit-1 cells.After 48 hours the Nit-1 cells transfected,the proteins are extracted from the cells, The expression of the myotrophin (MTPN) is analyzed by western blots and Immunocytochemical analysis. The level secretion of insulin in the Nit-lcell is measured by RAT/MOUSE INSULIN ELISA kit (Linco Research). The western blots and Immunocytochemical analyses revealed the protein of MTPN expressed decreasely. The level secretion of insulin in the Nit-lcell transfected with the plasmid of pAAV-MCS-miR-375 decreased 30% campared to the transfected with the plasmid of pAAV-MCS and the untransfected cells. We successly cloned the gene of the miR-375 and construct a plasmid of pAAV-MCS-miR-375 that produce the mature miR-375, which down-regulated the expression of the MTPN protein. Part II The study on Injure of pancreas induced by overexpression of miR-375The transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene was injected into the pronucleus of fertilized oocytes. Four transgenic founders were identified by PCR, Slot Blot and Southern Blot hybridization. We generated 17 transgenic mice in which the over-expression of miR-375 was under the control of a rat insulin promoter (RIP).The analysis of RT-PCR and real-time PCR of the expression of miR-375 revealed the expression of miR-375 in transgenic mice was 1.15 folds compare to the normal mice. We found the expression of the myotrophin (MTPN) in transgenic mice was 1/7.7 of the normal mice by real-time PCR and western blots analysis. The measure of the fasting serum insulin level of the transgenic mice showed the fasting serum insulin level of the female transgenic mice decreased significantly compared with the untreated control group (0.56144±0.091 vs0.8542±0.044, P<0.05), whereas the male are normal (0.8542±0.044vs0.8525±0.072). Body weight administered weekly, and the fasting blood glucose levels were measured with a portable glucose meter every two weeks. We found the female transgenic mice grown significantly slower than the untreated control group while the male grown normally and the fasting blood glucose was no significant difference among female transgenic mice, male transgenic mice and the untreated control group (2.98±0.57vs3.55±0.1721, 3.521±0.227vs3.55±0.1721). The expression of the gene relate to pancreas development showed PDX-1,INS1,ISL-1,PAX6,GLK,GLG,PP is up-regulated by RT-PCR analysis. The hematoxylin/eosin (H&E) analysis of the pancreas showed there was fatty degeneration in pancreas and the islet markedly shrinked. The hematoxylin/eosin (H&E) analysis of the lungs showed transgenic mice featured lymphocytic infiltration, bronchus inflammatory and bronchus putrescence. PartⅢPreliminary study on In Vitro Trans-differentiation of Non-β-cell into Insulin-producing cellsWe construct the plasmid of pAAV-MCS-PDX-1 and transfect 293T cell with it. After 48 hours transfected 293T cell with the plasmid of pAAV-MCS-PDX-1, RT-PCR analysis revealed that some genes relative to pancreatic development (PDX-1, ISL-land PAX6) were up-regulated; others (PAX4, Bate2 and PC2) were expressed whereas they are not expressed in untransfected 293T cells. But the expression of the insulin gene has not been detected yet. We also construct the plasmid of pAAV-MCS-Insulin, and transfected 293T cell with the the plasmid of pAAV-MCS-PDX-1 and pAAV-MCS-Insulin. The result showed PDX-1 boost up the expression of the insulin.