Effects On The Growth Of Human Cervical Cancer Cell Lines Induced By HPV16 Small Interfering RNA

ObjectiveTo investigate whether the growth of cervical cancer cell lines CaSki and Siha positive for human papillomavirus(HPV) 16 can be inhibited by HPV16 E6 small interfering RNAs (siRNAs) and HPV16 E7 siRNAs.MethodsThe experiment was divided into four groups: HPV16 E6 siRNA group, HPV16 E7 siRNA group, HPV16 E6+E7 siRNA group and control group. 1. RNAi was induced using synthetic siRNAs, which was transfected into CaSki cells and Siha cells by lipofectamine. The cell viability and the inhibition ratio of cell were measured using MTT. Flow cytometry(FCM) were performed to evaluate the apoptosis ratio and the distributions of cell cycle at various time points of post-transfection. 2. At 48 hours of post-transfection ,the expression level of p53,Rb mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR), and the level of these proteins were measured by Western Blotting .Result1 .The results showed that cells growth inhibition of E6 siRNA, E7 siRNA and E6+E7 siRNA were demonstrated and viability of cells was declined. FCM result showed that the apoptosis ratio of CaSki cells were 29.5﹪, 41.3﹪and 45.9﹪respectively, and that of Siha cells were 40.7%, 40.3% and 63.7% respectively at 48 hours post-transfection which were significantly higher than that of control group(p<0.05). Substantial G1 arrest was observed by FCM analysis.2. At 48 hours post-transfection,RT-PCR result showed that the level of p53 and Rb mRNA appeared unaffected compared to that of control (p>0.05).3.The basic expression of p53 and Rb protein was detected by Western blotting in control group. At 48 hours post-transfection, the expression of p53 and Rb protein in E6 siRNA group were upregulated compared to control group, and the expression of p53 protein is higher than that of Rb protein; the expression of p53 and Rb protein in E7 siRNA group were upregulated compared to control, and the expression of Rb protein is higher than p53 protein; the expression of p53 and Rb protein in E6+E7 siRNA group were higher than that of E6 siRNA and E7 siRNA alone in Siha cells, and the expression of p53 and Rb protein were not measured in CaSki cells.Conclusions1.HPV16 E6 siRNA can inhibit proliferation and induce apoptosis in cervical carcinoma cell lines CaSki and Siha. RNA interference techenology should provide a new rationale for specific gene-therapy approaches against HPV infection-related disease.2. E6 siRNA and E7 siRNA can upregulate the expression of Rb and p53 protein and reverse the function of Rb and p53 protein. The effect of E6+E7 siRNA is higher than that of E6 siRNA or E7 siRNA alone.