The Impact Of MiR-145on The Proliferation Of HepG2Cells

Objective: Occurrence and development of hepatocellular carcinoma development is aresult of multi-factor and multi-step synthesis. There has reached a consensus that it is acomplex polygenetic disease. Micrornas (miRNA), a present research hotspot in lifescience, has attracted extensive attention for it can serve a dual function of prompting andinhibiting cancer gene in development of hepatocellular carcinoma. Previous studiesshowed that miR-145is a kind of potential liver cancer "protective" miRNA, this studyaims to explore miR-145in human hepatocellular carcinoma HepG2cells, its relationshipwith liver cancer stem cells and the possible molecular mechanism, to clarify the possibleaction mechanism of miRNA in regulation of cell proliferation, to reveal the rule of livercancer cell growth, to explore a new way for the treatment of hepatocellular carcinoma.Methods（1）According to the conventional cultural method, culture human hepatocellularcarcinoma HepG2cells in vitro, select cells in logarithmic phase for experimental cells,vaccinate in the cell culture plate or culture bottle, when the cells are attached completely,discard the cell culture medium.（2）siRNA-NC-FAM and shRNA-GFP as transfection content respectively,Lipofectamine2000as transfection reagent, observe the transfection efficiency throughinverted fluorescence microscope, then do transfection efficiency evaluation.（3）Experiments in this paper are divided into the following four groups: mir-145analogtransfection group, analog negative control ontrast transfection group, control group onlywith transfection reagent and blank control group without any further processing.According to anthropogenic miR-145-5p gene sequence, design and synthetise doublechain simulation (miR-145-5p mimics). Package miR-145-5p mimics by Lipofectamine2000, and it gets into human liver HepG2cell lines. （4）Detect the expression level of miR-145-5p by real-time quantitative PCR.（5）48hours after transfection, to detect target gene OCT4protein expression level in thecells by Western blot.（6）Detect miR-145-5p’s promotive action to HepG2liver cancer cell lines growth atdifferent times (24h,48h,72h) by cell proliferation experiment method of CCK8.（7）Detect the expression changes of stem cell marker CD90mRNA by real-timequantitative PCR.Results:（1）Transfect HepG2cells with the medium dose of siRNAoligo is more effective.;（2）Contrast each of the four control groups, miR-145expression of miR-145analogtransfection group increases obviously.（3）miR-145overexpression inhibits the growth of human hepatocellular carcinomaHepG2cells;（4）48hours after transfection, miR-145overexpression of miR-145analog transfectiongroup can obviously reduce OCT4protein levels and the stem cell marker CD90mRNAexpression.Conclusion: MiR-145may inhibit the proliferation of HepG2cells by Cutting OCT4gene expression. miR-145is a kind of potential liver cancer "protective" miRNA.