Inhibition Of Human Papillomavirus 16 E6 Oncogene In Cervical Cancer By ShRNA Expression Vector

Objective:To research the specificity and efficiency of HPV16-E6 gene silence in CaSki and siha cells of cervical cancer in vitro and in vivo by siRNA Expression Vector. Methods:1. According to the computer aided design, 56nt oligonucleotide fragments containing different HPV16-E6 specific sequences were synthsized and cloned into the expression vector psiRNA, and three recombinant vectors E6-1, E6-2 andE6-3 were constructed.2. The vectors were transfected into CaSki cells and SiHa cells with liposomes. Stable shRNA expressing cell lines were obtained by G418 selection.The transcription of HPV16-E6 mRNA was detected with FQ-PCR (fluorescence quantitative PCR)and the Expression of protein and Apoptosis was detected with flow cytometry at different times after transfection.3. the changes of biological behavior were monitored by cell growth curves and cloning efficiency.4. Human cervical cancer CaSki cells were transfected by vector E6-2 and PO were seeded hypopercutaneously on nude mice, tumor volume were measured, the expression of the HPV16-E6 protein were monitored by IHC, the apoptosis of tumor cells were detected by TUNEL.Results:1. HPV16-E6-specific siRNA expression vectors were confirmed by PCR analysis and DNA sequencing.2. HPV16-E6 mRNA expression could be inhibited 81.41%, 89.57%, 71.24% respectively by vector E6-K E6-2 and E6-3 group after the formation of CaSki cellular positive colonies and the inhibition rates of HPV16-E6 protein of E6-1, E6-2 and E6-3 group were 82.1 %, 98.1 % , 89.4% respectively. So the inhibition effect of siRNA expression vector E6-2 was more effective.3. The apoptotic rates induced by E6-1, E6-2 and E6-3 were 2.9%, 15.1%, 12.6%respectively after the formation of CaSki cellular positive colonies. So siRNA expression vector E6-2 could induce apoptosis more effectively.4. HPV16-E6 mRNA expression could be inhibited 20.48 %, 12.13%, 28.05 % respectively by vector E6-1 , E6-2 and E6-3 after the formation of siha cellular positive colonies and the inhibition rates of HPV16-E6 protein of E6-1, E6-2 and E6-3 were 24.61 %, 49.96%, 40.01% respectively. The expression level of HPV16-E6 mRNA and protein were not varied significantly.5. HPV16-E6 mRNA expression could be inhibited 89.57%, 70.73 %,65.89 %,51.75%,9.95% respectively by vector E6-2 at 0w,2w,4w,6w,8w after the formation of CaSki cellular positive colonies, and the inhibition rates of HPV16-E6 protein were 98.1 % , 87.6 % ,81.3 % ,72.5 % ,25.5% respectively.The apoptotic rates were 15.1%, 11.5 %, 7.7% , 1.7%, 1.9 %of E6-2 group respectively at 0w,2w,4w,6w,8w after the formation of CaSki cellular positive colonies.6. After transfected by vector PO, The expression level of HPV16-E6 mRNA and protein were not varied significantly at any time.7. The proliferation and cloning efficiency of caski cell could be inhibited significantly by vector E6-2. After transfected by vector PO, cell proliferation were not changed significantly.8. In vivo tests, the growth rates of tumor were markedly inhibited by vector E6-2. The expressions of HPV16-E6 protein were inhibited and the apoptosis of tumor cells increased significantly.