Sidrosha A Cell Line Stability And Mir491-5 P Induced Pancreatic Cancer Cell Apoptosis Mechanism Research

MicroRNA (miRNA) is a class of small non-coding RNAs with21-25nt in length functioning posttranscriptionally to modulate targeted gene expression. MicroRNAs can modulate gene expression by inducing mRNA degradation or inhibiting mRNA translation. So far650miRNAs have been identified in the human genome. About30%of human protein expression can be regulated by these miRNAs. MicroRNAs influence diverse cellular processes ranging from cellular differentiation, proliferation, apoptosis and metabolism to cancer. Because of the important features of the miRNAs, the research on the miRNAs has become one of the hottest scientific research fields in recent years. The more detailed functions of miRNAs will be gradually revealed by people through continuous exploration.Drosha play a very important role in the biosynthesis of miRNAs as a member of the RNaseⅢ family, it mediated the procession from pri-miRNA to-70nt pre-miRNAs. In cells the Drosha gene mutations will affect the normal biosynthesis of miRNAs, so the model of the Drosha gene mutation will help to study the functions of some miRNAs.In the first part of this thesis, we constructed Drosha-1deficient293stable cells (293-siDrosha) and studied microRNA processing by using the established293stable cells. Study as follows:1) the pSicoR-human Droshal plasmid DNAs were transfected into293cells and selected for neomycin resistant clones with1200μg/ml G418for2-3weeks;2) the mRNA and protein expressions of Drosha were detected by RT-PCR, real-time PCR and Western blotting;3) the expressions of hsa-miR-491-5p in293cells, uncloned293-siDrosha cells (293-siDrosha mixed) and the cloned293-siDrosha cells (293-siDrosha#3cells) were detected with poly(A) real-time RT-PCR. The experimental results show that compared with the control cells, the mRNA and protein expressions of human Droshal in cloned293-siDrosha cells were dramatically decreased. The expression levels of hsa-miR-491-5p in the cloned293-siDrosha cells have about120fold reduction as compared with that of the control cells. In this experiment,293-siDrosha stable cells were successfully constructed. The cloned293-siDrosha cells may be useful as a model system for the study of microRNA functions.miRNA can regulate human growth and development, physiological and pathological state, therefore the miRNA in the body can be potentially served as a new and effective biomarker for the evaluation of human physiology and disease state. Studies have shown that miRNA expression are different between normal tissue and pathological tissue, even if in the same organization, the miRNA expression is not the same in the different period of differentiation. This different expression of the tumor-specific miRNA can help diagnosis the origin and type of specific tumors. More and more evidence support that the miRNA can be used as biomarker of human tumors. In the normal cells the miRNA abnormal expression may induce cancer and other diseases, so the study on the regulation of miRNA in normal and cancer cells may provide new biomarker for diagnosis and treatment of human diseases.Pancreatic cancer is a type of aggressive disease that causes high mortality in human population without early signs of symptoms. Biomarker screening for pancreatic cancer is important for early diagnosis and prognosis of the disease and has been paid a great attention in recent years.In the second part of this thesis the expression profiles of miR491-5p was analyzed and determined. We found that miR491-5p has extremely high level of expression in normal pancreatic tissue versus pancreatic cancer cells. We hypothesized that miR491-5p may have a potentially important role in the process of pancreatic cancer. In this study, we transiently transfected the chemically synthesized miR491-5p and a retroviral vector derived miR491-5p (pLNCX2-G491) into SW1990cells and test their effect in SW1990cells. Study as follows:1) three computer programs (DIANA-MICROT, MICRORNA and TARGETSCAN) were employed to predict the potential targeted genes for mR491-5p. We are looking at these targeted genes that were predicted by at least two different soft-wares. Further restriction was emphasized on the targeted site(s) being potentially associated with pancreatic cancer development. The prediction of the potential targeted genes for miR491-5p were confirmed by Western blotting、RT-PCR、 qRT-PCR;2) we transiently transfected the chemically synthesized miR491-5p and pLNCX2-G491into SW1990cells, the effect of miR491-5p on the proliferation and apoptosis of pancreatic cancer SW1990cells were evaluated by cell-counting kit-8(CCK-8) and flow cytometric analysis;3) the signaling pathways of miR491-5p in pancreatic cancer cells were detected by western blotting. The experimental results as follows:1) TP53and Bcl-xL were predicted to be the most likely targeted gene by mR491-5p in pancreatic cancer;2) miR491-5p effectively downregulated TP53and Bcl-xL gene expressions in pancreatic cancer cells SW1990;3) overexpression of miR491-5p inhibited cell proliferation and promoted cell apoptosis in SW1990cells;4) miR491-5p effectively activated intrinsic mitochondrial apoptotic pathway and miR491-5p inhibited both STAT3and PI3K/Akt signaling pathways.Thus, our data identified a miR491-5p/Bcl-xL/TP53axis functioning in the induction of apoptosis in pancreatic cancer cells. Current study also provides the valuable information for potential biomarker identification that might be beneficial for future diagnosis and treatment of human pancreatic cancer.