The Influence Of HPV16E6 Gene Stable Silencing On The Promoter Hypermethylation Status Of E-cadherin In SiHa Cervical Cancer Cell Lines.

Objective: Cervical cancer is the most common gynecologic malignant tumor in our country, and is the constant threat to women's life and health.With the development of medical research, the human papillomavirus type 16(HPV16) is considered to be the main exogenous pathogenic factors of cervical cancer, the E6 gene is most important in the inhibition of cell cycle,interfering with the chromosome stability,impacting signal path,and immune escaping.E-cadherin is the main molecular in epithelial cell adhesion connection.When silence this gene,the epithelial cell phenotype and the invasive ability is changed.Our previous experiments have confirmed that high expression of HPV16E6 protein in cervical squamous carcinoma tissues is related to the methylation of E-cadherin gene promoter region Cp G island and the low expression of E-cadherin protein.So,what is the cervical cancer pathogenesis caused by HPV? Whether through the E-cadherin methylation?The question is still to be solved.This study is to construct a lentivirus sh RNA expression vector,and to screen cell lines for silencing the expression of HPV16E6.We compared the E-cadherin expression and methylation status after silencing HPV gene,also we analysis the correlational biological function.We preliminary anlysis the mechanism that HPV regulate the E-cadherin methylation.In order to find valuable targets for gene therapy of cervical cancer.Methods: 1.We cultivate the cervical cancer Si Ha cell lines.at the same time,we construct a lentivirus sh RNA expression vector,then transfect into the competent cells for producing positive clone and sequencing. We pack this vectors by co-transfecting into 293 T cells,and detect the viral titer.Then we screen the stable transfection cell lines by using puromycin.2.We divided into three groups, which is sh RNA E6 group, Empty vector group,Blank control group.The m RNA level of HPV16E6 and E-cadherin are detected by RT-q PCR.The protein expression level of E-cadherin are used by Western Blot.Then,we observe the change of the cell proliferation and migration of the Si Ha cells in different groups which is used by CCK-8 method and the Wound Healing experiment.3.Methylation specific PCR(MSP) method is used to detect the methylation status of E-cadherin gene promoter Cp G island region in Si Ha cells.Results: 1.We have successfully constructed the lentivirus sh RNA vector.Under the fluorescence microscope,we can see green fluorescence.The viral titer is 5×108TU/ml.After the stable screening,the HPV16E6 m RNA level in sh RNA E6 group is abviously lower than the other group(P<0.01).It is proved that we have screened the stable silencing cell lines successfully.2.In sh RNA E6 group,the expression of E-cadherin m RNA and protein were significantly higher than that in the blank vector group and blank control group(P<0.01), and the difference between the blank vector group and blank control group was not statistically significant(P>0.05).At the same time,the cell proliferation and migration of the Si Ha cells were obviously decline than the other groups(P<0.05).3.After Knocking down HPV16E6 gene, the unmethylation amplification was positive,whereas the intensity of the amplified methylation products was significantly weaker than that in the negative vector group and blank control group, while the methylation amplification was positive in the blank vector group and blank control group.Conclusion: 1.The lentivirus sh RNA expression vector could stable silence HPV16E6 gene successfully.2.Knocking down the HPV16E6 gene increased the expression of E-cadherin in cervical cancer Si Ha cell lines,and decreased the ability of cell proliferation and migrationso,as to decreased the level of E-cadherin promoter methylation.To a certain extent,it partly reversed the methylation status of E-cadherin promoter, and caused E-cadherin to be re-expressed.