Preliminary Study Of Molecular Pathologic Mechanism In Abnormal Expression Of P16 In Squamous Carcinoma

Cervical cancer is one of the most common female malignancies,its morbidity ranks second in female malignancies which is only second to breast cancer,China is a high-incidence area of cervical cancer.A large number of studies have found that high-risk HPV infection is a necessary condition for the occurrence of uterine cancer,p16 expression in HPV-positive cervical intraepithelial neoplasia and squamous cell carcinoma is significantly higher than normal cervical epithelium,but molecular mechanisms of its abnormal expression and significance is unknown.In this study,Caski cells in HPV16-positive cervical squamous cancer and HCC94 cells in HPV-negative well-differentiated cervical squamous cancer were used to study the molecular mechanism of p16 elevated expression in cervical squamous cancer.Firstly,HPV16 specific primers E6/E7 were designed,then the E6/ E7 gene was amplified by RT-PCR to construct E6/E7 overexpressing plasmid pc DNA3.1-HPV16E67.The HPV 16 E6 / E7 gene overexpressing plasmid and control plasmid were transfected into HCC94 cells by lipofectamine,real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of E6/E7 and p16^ink4a in HCC94 cells.E6/E7 gene specific shRNA interference plasmid was designed and transfected into Caski cells with empty plasmid vector by lipofectamine-mediated plasmids,real-time fluorescent quantitative PCR and Western blotting were used to detect E6/E7 gene silencing efficiency as well as m RNA and protein expression levels of p16^ink4a.The results showed that in E6/E7 overexpressing HCC94 cells the mRNA and protein expression levels of E6/E7 and p16^ink4a significantly increased;in E6 / E7 silencing Caski cells,the mRNA and protein expression quantity of E6/E7 and p16^ink4a significantly decreased.We further explored the effect of overexpression and silencing of E6 and E7 gene alone on the expression of p16^ink4a gene.E7 overexpression plasmid,E6 overexpression plasmid and E6,E7 interfering RNA were constructed（assisted by Tianyihuiyuan corporation）.The E6,E7 overexpression plasmids and empty plasmid vectors were transfected into HCC94 cells by means of lipofectamine.Real-time quantitative PCR was used to detect the expression of mRNA of E6,E7 and p16^ink4a in three groups of HCC94 cells.Western blotting was utilized to detect the expression of protein of E6,E7 and p16^ink4a in three groups of HCC94 cells.E6,E7-specific siRNA and negative control RNA were transfected into caski cells respectively.The expression of mRNA of E6,E7 and p16^ink4a in three groups of caski cells were detected by Real-time fluorescence quantitative PCR.The expression of protein of E6,E7 and p16^ink4a in three groups of caski cells were detected by Western blotting.The results demonstrated that the control group and the group that individually overexpress and silence E6 gene did not lead to the change of the expression of p16^ink4a gene in HCC94 and caski cells.The group that overexpress E7 gene caused the increased expression of p16^ink4a gene in HCC94 cells and the decreased expression of p16^ink4a gene in caski cells.In order to further study the regulation of p16^ink4a gene on cycle related genes（cyclinD1）and proliferation-related gene Ki67 in human cervical squamous cell carcinoma lines and the effect of p16^ink4a gene on cell growth curve.The overexpression plasmid and interfering RNA of p16^ink4a gene were constructed.The p16^ink4a gene overexpression plasmid and empty plasmid were transfected into the cervical squamous cell carcinoma cell line HCC94 through lipofectamine.The expression of mRNA and protein of p16^ink4a,cyclinD1 and Ki67 in two groups of HCC94 cells were detected with Real-time quantitative PCR and Western blotting.And MTT assay was used to detect the level of proliferation in two groups of HCC94 cells.The p16-siRNA and negative control siRNA were transfected into caski cells by lipofectamine respectively.Real-time fluorescent quantitative PCR and Western blotting were utilized to detect the silencing efficiency of p16^ink4a gene and the expression of mRNA and protein of cyclinD1 and Ki67 in two groups of caski cells.The level of proliferation in two groups of caski cells were detected by MTT assay.The results showed that the overexpression of p16^ink4a could induce the increased expression of cyclinD1 and Ki67 and there is a upward trend in the cell growth curve.The silencing of p16^ink4a could decrease the expression of cyclinD1 and Ki67,and the cell growth curve showed a decreasing trend.The level of p16^ink4a gene in human cervical squamous cell carcinoma was positively correlated with cyclinD1 and Ki67 gene.The overexpression of p16^ink4a promoted the proliferation of human cervical squamous cell carcinoma.In this study,we further discussed and elucidated that the abnormal expression of p16^ink4a gen in HPV-associated cervical intraepithelial neoplasia and squamous cell carcinoma is associated with viral oncogene,it is especially related to E7 gene.As tumor suppressor gene,P16ink4 a doesn’t play a role in carcinostasis in cervical cancer,and it may upregulate the expression of cyclinD1 and Ki67,promote the conversion of cell cycle and cell proliferation,and involve in the evolution process from cervical intraepithelial neoplasia to squamous cell carcinoma,which laid the foundation for the further study of the mechanism of p16^ink4a gene in cervical disease.