The Study On Relationship Between The Expression Of MiR-221、miR-222、miR-92b And P57^kip2 In HepG2Hepatoma Cells

Objective:In order to clarify the regulation relationship between microRNA (miRNA) and the expression of P57kip2protein, we transfect the inhibitors of miR-221、miR-222and miR-92b which associating with p57kip2in HepG2cells (HCC-derived cell lines) to inhibit the expression of the three miRNAs, and then observe the change of P57kip2protein and cell viability after transfection, and to explore the upstream modulation mechanism of p57kip gene and its role in hepatocarcinogenesis from the cell and protein levels.Methods:Human HepG2hepatoma cells and QSG-7701hepatocytes were cultured and treated differently in vitro,the expression of miR-221, miR-222and miR-92b were detected in both of the two cells using quantitative real-time PCR (qRT-PCR). And the HepG2cells were randomly divided into following groups, including the blank control, the negative control group and the miRNA inhibitors transfected group. Through the lipo2000, which is a kind of transfection reagent, we transfected the miR-221inhibitor the miR-222inhibitor and the miR-92b inhibitor into HepG2hepatoma cells.48hours later, the p57kip2protein were detected by western blotting, and Using4-methyl-triazole blue colorimetric method (MTT) for detection of miRNA inhibitor on HepG2cell viability effects.Results:1.QRT-PCR:miR-221、miR-222、miR-92b were expressed in HepG2hepatoma cells, but compared with QSG-7701hepatocytes, the expression level of the three miRNAs are downregulated.There were statistically significance in the expression of the three miRNAs between QSG-7701hepatocytes and HepG2hepatoma cells:miR-221:p=0.035<0.05; miR-222: p=0.021<0.05; miR-92b:p=0.016<0.05.2. The transfection efficiency in HepG2hepatoma cells were all obove90%when transfected the fluorescent labeled siRNA transfection control after transfection5hours,24hours,48hours and72hours.3. MTT:Compared with the blank control group, the HepG2hepatoma cell viability of the negative control group are no significant effects, but the miR-221inhibitor transefected group, the miR-222inhibitor transefected group and miR-92b inhibitors transefected group were reduced. There was no statistically significance in the cell viability between the blank control group and the negative control group (p=0.074>0.05). But there were statistically significance between the miR-221inhibitor transefected group and the negative control group (p=0.003<0.01), the miR-222inhibitor transefected group and the negative control group (p=0.002<0.01), the miR-92b inhibitor transefected group and the negative control group (p=0.001<0.01).4. Western blotting:Compared with the blank control group, the expression level of p57kip2protein in the negative control group are no significant effects. And the expression level of p57kip2protein in the miR-221inhibitor transefected group, the miR-222inhibitor transefected group and the miR-92b inhibitors transefected group were higer than the negative control group. There was no statistically significance in the expression of P57kip2protein between the blank control group and the negative control group (p=0.346>0.05). And there were statistically significance in the expression of P57kip2protein between the miR-221inhibitor transefected group and the negative control group (p=0.004<0.01), the miR-222inhibitor transefected group and the negative control group (p=0.006<0.01), the miR-92b inhibitor transefected group and the negative control group (p=0.034<0.05).Conclusions:1. miR-221.. miR-222and miR-92b were expressed in HepG2hepatoma cells2. p57kip2should be the target gene of miR-221, miR-222and miR-92b in hepatocellular carcinoma, the three kinds of miRNAs negatively regulate the expression of P57kip2protein and promote cell proliferation.