Regulatory Effect Of High-risk HPV E6 And E7 Proteins On Hippo Tumor Suppressor Pathway

Objective: To construct miRNA interference vectors targeting HPV16E6,E7 and HPV18E6 and E7 respectively,then to transfect SiHa(HPV16 positive squamous cell carcinoma)cells and HeLa(HPV18 positive adenocarcinoma)cells,silence E6 and E7 oncogenes,and observe the effects of inhibiting E6 and E7 genes on the expression of Yap(major effector of Hippo signaling pathway),E-cadherin and N-cadherin(epithelial-mesenchymal transformation related genes).To explore the underlying mechanism of HPV E6 and E7 protein regulating Hippo signaling pathway,and to analyze the correlation between HPV and epithelial-mesenchymal transition(EMT)in cervical cancer cells.Methods:1.SiHa and HeLa cells were cultured in vitro.2.MiRNA interference plasmids against HPV16E6,HPV16E7,HPV18E6,HPV18E7 and a negative control microRNA interference plasmids were designed respectively.3.SiHa and HeLa cells were transfected by liposome transient transfection technology,and the plasmids with high silencing efficiency were selected for subsequent stable transfection.4.The stable transfected cell lines of HPV16,HPV18E6 and E7 genes were constructed respectively.5.Real-Time PCR method was used to detect the expression of Yap,E-cadherin and N-cadherin before and after transfection.Results:1.Inverted phase contrast microscopy showed that SiHa and HeLa cells grew vigorously and their cell morphology showed diversification.2.Successfully constructed and transfected plasmid: For SiHa cells,the expression of HPV16E6 mRNA was significantly decreased after transfection of plasmids HPV16E6-miRNA,and the Real-Time PCR results showed that the effective inhibition rate was 77.6%.The HPV16E7-miRNA2 and miRNA4 could effectively inhibit the expression of HPV16E7 gene and the effective inhibition rates were 67.2% and 59.3% respectively.Therefore,in the SiHa group,stable transfected cell lines were constructed with plasmids HPV16E6-miRNA and HPV16E7-miRNA2.For HeLa cell line,the expression of HPV18E6 mRNA was significantly decreased after transfection of plasmid 18E6-miRNA,and Real-Time PCR showed that the effective inhibition rate was 87.5%.Plasmids HPV18E7-miRNA1 and miRNA4 could effectively inhibit the expression of HPV18E7 mRNA and the effective inhibition rates were 68% and 45.6% respectively.Therefore,in HeLa group,the stable transfected cell lines constructed with plasmids 18E6-miRNA and18E7-miRNA1 were used for subsequent experiments.3.Real-Time PCR results showed that after HPVE6 and E7 gene expression was inhibited,the expression of Yap gene was significantlyreduced,and the expression of EMT marker N-cadherin was significantly decreased,while the expression of E-cadherin was significantly increased.Conclusion:HPV E6 and E7 can promote EMT in cervical cancer cells.HPV E6 and E7 may promote EMT process by regulating Hippo signaling pathway.