Research On The Relationship Between The Expression Of MiRNA-221、miRNA-222、miRNA-92b And P57^kip2 MRNA In HepG2Hepatoma Cell Line

Objective:In order to study the relationship between the expression of miRNA-221、 miRNA-222、miRNA-92b and p57kip2mRNA in HepG2hepatoma cell lines and further clarify the regulatory mechanism of p57kip2gene upstream as well as the role in the hepatocarcinogenesis. This paper intends to detect three miRNAs regulation of relation with p57kip2mRNA and explore unknown areas of liver cancer as well as provide a new basis in cell and molecular leve for clinical diagnosis and treatment of liver cancer.Methods:HepG2cells and QSG-7701cells were cultured in vitro and detected miRNA-221、miRNA-222、miRNA-92b expression by real time quantitative PCR in both cells. Using respectively three miRNA inhibitors transfected HepG2hepatoma cell lines,fluorescent labeled siRNA evaluating transfection efficiency. Transfection after24hours detecting cell activity by MTT, detecting p57kip2mRNA expression by RT-PCR and comparing to p57kip2mRNA, analyzing the relationship between the three miRNAs and p57kip2mRNA expression.Results:(1) Fluorescence quantitative PCR results:miRNA-221, miRNA-222and miRNA-92b are expressed in HepG2cells,but expressions were lower than QSG-7701cells.miRNA-221:t=-3.145, p=0.035<0.05, the difference was statistically significant; miRNA-222:t=-6.875, p=0.021<0.05, the difference was statistically significant; miRNA-92b:t=-7.722, p=0.016<0.05, the difference was statistically significant.(2)Transfection efficiency of the test results:fluorescently labeled siRNA was transfected into HepG2cells in the transfection efficiency of the four time periods in more than90%.(3) MTT assay results:Three miRNA inhibitors were transfected into HepG2cells and detected obviously reducing the cell activity after24h transfection. Blank control group compared with negative control group, p=0.546>0.05, the difference was not statistically significant;miRNA-221compared with negative control group, p=0<0.01,the difference was statistically significant; miRNA-222compared with negative control group, p=0<0.01, the difference was statistically significant;miRNA-92b compared with negative control group,p=0<0.01, the difference was statistically significant.(4) RT-PCR results:the experimental group (transfected miRNA-221.. miRNA-222, miRNA-92b inhibitor), the negative control group (transfected similar miRNA inhibitors siRNA),the expression p57kip2mRNA levels of the experimental groups higher than the negative control group.Blank control group compared with negative control group, p=0.565>0.05, the difference was not statistically significant;miRNA-221compared with negative control group, p=0.044<0.05, the difference was statistically significant; miRNA-222compared with negative control group, p=0.042<0.05, the difference was statistically significant; miRNA-92b compared with negative control group, p=0.012<0.05, the difference was statistically significant.Conclusions:(1) miRNA-221, miRNA-222, miRNA-92b three kinds of miRNA expression both in HepG2cells and QSG-7701cells.(2) p57kip2should be the target gene of the miRNA-221、miRNA-222and miRNA-92b in hepatocarcinoma. These three kinds of miRNAs are negatively regulated the p57kip2mRNA expression, promoted cell proliferation and may be related to the occurrence of liver cancer.