Construction Of Eukaryotic Expression Vector Of SiRNA Specific For Targeting PCNA Gene And Its Effects On HepG2 Cell Line

Objective:To construct eukaryotic expression vector of siRNA specific for targeting PCNA gene and transfected into hepatocelluar carcinoma HepG2 cells,to initially investigate the effect of recombinant plasmid on the proliferating inhibition and biological effect on HepG2 cells,to provide the experimental evidence and a new tool to further explore the function of PCNA gene and the feasibility of its gene therapy.Methods:In the early stage of our work,four in vitro synthesized double-stand siRNA (dsRNA)were respectively transfected into three types of digestive carcinoma cell lines by Lipofectamine^TM 2000 reagent.No.2 PCNA siRNA which showed the most effective inhibition of tumor cell growth among the four candidate siRNAs.According to the No.2 PCNA siRNA sequence,we converted No.2 PCNA siRNA into cDNA coding expression of shRNA(small hairpin RNAs).The cDNA was synthesized and inserted into plasmid pSilencer2.0-U6 to construction of eukaryotic expression vector of siRNA specific for targeting PCNA gene,and identified by the sequence analysis.The plasmid was transiently transfected into hepatocelluar carcinoma HepG2 cells for 36 hours by Lipofectamine^TM 2000, the uptake rates of pEGFP(expression green fluorescence protein plasmid) on HepG2 cells were tested by flow cytometry,while the morphological expression of pEGFP was observed under fluorescence microscope.The plasmid was transiently transfected into hepatocelluar carcinoma HepG2 cells for 48 hours,proliferation and the clone formation of HepG2 cells were detected by CCK-8 and clone formation assay respectively,the morphlogy was examined by microscope.The percentage of hypodiploid cells of HepG2 cells treated with pShPCNA for 48h was determined with propidium iodide(PI) staining by flow cytometry. Early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry. The morphology was examined by fluorescence microscope after hoechst 33258 staining.Results:The pShPCNA of recombinant plasmid identificated by the sequence analysis completely coincided with the designs.The recombinant plasmid was transiently transfected into HepG2 cells by Lipofectamine^TM 2000.Compared with control group and negative control group,the proliferation and clone formation of HepG2 cells treated with pShPCNA for 48 h were significantly inhibited(P＜0.01).Percentages of hypodiploid cells and early apoptosis rates were significantly higher in treatment groups than those in control and negative control group(P＜0.01).Conclusion:The siRNA eukaryotic expression vertor targeting PCNA mRNA has been successfully constructed,and effectively inhibits proliferation and induces apoptosis on HepG2 cells.