The Study On The Effect Of HPV16 E6 ShRNA And Nanog ShRNA In SiHa Cell

Objectives:Cervical carcinoma is one of the most common gynecological malignancies,with more than 520000 new cases reported annually,and approximately 12% of these cases occur in China.Study sees more cervical cancer at young age and the incidence of cervical cancer have increased slightly.It is well known that persistent infection of human papillomavirus(HPV)is causal event in cervical carcinogenesis,70% of cervical cancer patients were found to be infected with either HPV16 or HPV18.The main pathogenesis of cervical cancer is that High-risk HPV infects the host cells persistently,HPV DNA are integration into the host genome,HPV oncogenes E6/E7 express,which makes the cells malignant transformation and have the ability of tumorigenic,invasion and metastasis.Besides,there are additional genetic factors contribute to the progression to cervical cancer.Emerging evidences suggest that Nanog is involved in cervical tumorigenesis.Down regulation of Nanog inhibits the proliferation and migration of tumor cells,strengthens chemoresistance cervical cancer cells.Both E6 and Nanog present high expression in cervical cancer tissue,and their expression levels showed significantly differences according to different cell growth,chemoresistance,metastasis,and immune evasion.However,the carcinogenic mechanism of E6 and Nanog in cervical cancer cells remains unclear.We aimed to explore the role of HPV E6?Nanog and the interaction between them in the carcinogenesis and progression of cervical cancer,and to suppose molecular mechanism,and to assess the value of shRNA in cervical cancer treatment.Methods: 1.According to siRNA design principles,six interference sequences and a negative control sequence which were no homology with any other human genes were designed and seven recombinant plasmid vectors of shRNA were established.Transfect those into SiHa cell by proliferation.To detect the inhibition efficiency by RT-PCR and select out the most efficient recombinant plasmid.2.Recombinant plasmid shRNA-E6-3 and shRNA-Nanog-3 were transfected into the cervical cancer SiHa cells by proliferation.RT-PCR was performed to detect the mRNA levels of E6 and Nanog in SiHa cells.3.By western blot,the expression of proteins p53,Nanog,STAT3,Cyclin D1 in SiHa cell were investigated.4.The growth rate of SiHa cell treated with shRNA was determined with CCK8 assay.Transwell assay be used to observe migration capacity of Si Ha cells.5.Flow cytometery were conducted to examine the SiHa cell apoptosis and cell cycle after transfection of E6 shRNA and Nanog shRNA.6.SPSS16.0 and MS Excel were used to statistically analyze the experimental data.Significance level: P<0.05.Results: 1.Seven shRNA plasmids were successfully constructed(shRNA-E6-1,shRNA-E6-2,shRNA-E6-3,shRNA-Nanog-1,shRNA-Nanog-2,shRNA-Nanog-3 and shRNA-Control,respectively).2.In SiHa cells,quantitative real-time PCR showed that E6 expression reduced by shRNA-E6-1,shRNA-E6-2 and shRNA-E6-3 were 33.16%,35.13% and 52.53% and Nanog expression reduced by shRNA-Nanog-1,shRNA-Nanog-2,shRNA-Nanog-3 were 53.67%,54.50% and 56.78%,respectively.3.E6 and Nanog mRNA can be silenced by shRNA effectively.Both shRNA-E6 and shRNA-Nanog can decrease expression ofNanog,STAT3 and Cyclin D1 in SiHa cell.4.It was confirmed by CCK8 assay that shRNA-E6 and shRNA-Nanog can significantly inhibit the proliferation of SiHa cell(P<0.05).By Transwell assay,cell migration ability were weaken(P<0.05).5.After transfection of shRNA-E6 and shRNA-Nanog,cell cycles and apoptosis of SiHa cell can also be altered;shRNA-E6 and sh RNA-Nanog can significantly induce cells to arrest at the G0/G1 phase,delay progression of the cell cycle and decrease cell proliferation compared with shRNA-Control.Conclusion:HPV16 E6 shRNA and Nanog shRNA recombinant plasmid expression vector were successfully constructed.The recombinant plasmids can silence the expression of HPV16 E6 and Nanog genes effectively,resulting in the expression of Nanog? STAT3?Cyclin D1 decrease,and cell growth inhibition,migration ability weaken,and made the cell cycle asset in G0/G1 stage in SiHa cells.Treating SiHa cell with co-transfection of E6 shRNA and Nanog shRNA can significantly induce cells to arrest at the G0/G1 phase,delay progression of the cell cycle and decrease cell proliferation in coordination.