Effect Of HBx-shRNA And H-CCL20 On HepG2.2.15 Cells

Research objectiveTransfection into HepG2.2.15 cells by constructing pGenesil-3.1-HBx-shRNA vector and pHBLV-h-CCL20 vector,to investigate the effects of HBx silencing and h-CCL20 overexpression on the growth of HepG2.2.15 cells,in order to clarify the molecular mechanism of the two genes involved in the development of liver malignancy.Research methods(1)A pGenesil-3.1-HBx-shRNA vector was constructed and HepG2.2.15 cells were transfected by electroporation.(2)Real-time fluorescent quantitative PCR was used to detect the difference of HBx and h-CCL20 expression levels;The cell proliferation activity was detected by CCK8 assay;Detection of HBsAg and HBeAg in the supernatant of each group of cells;Flow cytometry was used to detect the apoptosis of HepG2.2.15 cells.(3)Construction of pHBLV-h-CCL20 Vector and transfection of HepG2.2.15 Cells,obvious green fluorescence was observed under a fluorescence microscope,it was demonstrated that the pHBLV-h-CCL20 vector was successfully transfected into HepG2.2.15 cells.Research result(1)The pGenesil-3.1-HBx-shRNA vector and the pHBLV-h-CCL20 vector were successfully constructed and successfully expressed in HepG2.2.15 cells.(2)From the results of real-time fluorescence quantitative PCR,the HBx gene expression of HepG2.2.15 cells that had been successfully transfected with the pGenesil-3.1-HBx-shRNA vector was reduced by 28% compared to HepG2.2.15 cellstransfected with the empty vector,compared with the control group,the difference was statistically significant(P<0.05),indicating that shRNA inhibited the expression of HBx gene.(3)CCK-8 assay showed that the proliferation of HepG2.2.15 cells was significantly inhibited by shRNA and the cell proliferation was decreased by 47%.there was a significant difference compared with the control group(P<0.05).(4)After transfected with pGenesil-3.1-HBx-shRNA vector,the expression of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased significantly,and the apoptosis of HepG2.2.15 cells increased significantly,there was significant difference compared with the control group(P<0.01),thus indicating that the HBx gene plays a role in apoptosis.(5)The pHBLV-h-CCL20 vector overexpressed the h-CCL20 gene in HepG2.2.15 cells,and the overexpression of h-CCL20 gene promoted the apoptosis of HepG2.2.15 cells.Conclusion(1)RNAi technology was used to transfect cells by electroporation in HepG2.2.15 cells,the expression of HBx gene was inhibited,the expression of HBsAg and HBeAg in the supernatant was decreased,and the proliferation of HepG2.2.15 cells was inhibited,promote cell apoptosis.(2)The pHBLV-h-CCL20 vector overexpressed h-CCL20 gene in HepG2.2.15 cells,and the overexpression of h-CCL20 gene promoted the apoptosis of HepG2.2.15 cells,providing a theoretical basis for the study of h-CCL20 gene.