Role Of MUC16in The Development And Progression Of HPV16-Related Cervical Carcinoma

Background The sustained expression of high risk-human papillomaviruses (HR-HPVs)(such as HPV16) E6oncogenes is the most important risk factors lead to disease.Short hairpin RNA (shRNA) targeting HPV16E6was designed and screened respectively. And the silencing efficiency was more than95%. Microarray analysis was dectcted by Agilent human genome. It was found that mucin antigen16(MUC16) gene was down-regulated (2-5times) after silencing the expression of E6. Whether MUC16is the targets of HPV16and the direct regulation mechanism on E6are unclear. Therefore, there will be more important significance to study the relationship between the expression of E6and MUC16in-depth for exploring new treatments for HPV16-associated tumors.Objectives Taken HPV16E6positive and negative cervical carcinoma cells lines, normal cervical squamous epithelium, cervical intraepithelial neoplasias(CIN) and cervical squamous carcinoma tissues as the research object to analysis the relationship of HPV16E6and MUC16from cells and tissues levels. It intends to clarify the role of MUC16in the development and evolution of HPV16-related cervical carcinoma and enrich the carcinogenic mechanisms of HPV16E6.Methods1. Taken HPV16E6positive(CaSki,SiHa) and negative(C-33A) cervical carcinoma cells as the research object, the expression of HPV16E6and MUC16were detected by Western blotting, and analyze the relationship between HPV16E6and MUC16.2. Taken HPV16E6positive and negative normal cervical squamous epithelium, CIN and cervical squamous carcinoma tissues as the research object, the expression of HPV16E6and MUC16were detected by immunohistochemistry, and analyze the relationship between HPV16E6and MUC16.3. By gene recombination technology, the recombinant plasmid pGenesil-1which expressed short hairpin RNA (shRNA) targeting MUC16gene (named pGenesil-1-shRNA-MUC16) and the control plasmid pGenesil-1which expressed unrelated sequence (named pGenesil-1-shRNA-vect) were constructed.4. PGenesil-1-shRNA-MUC16plasmid and pGenesil-1-shRNA-vect plasmid were transfected into CaSki cells with liposomes. Obtained the G418resistant cells by G418screening (named CaSki-shRNA-MUC16cells, CaSki-shRNA-vect cells). MUC16silencing efficiency is identified by RT-PCR and Western blotting.5. The apoptosis, migration and invasion of CaSki cells after silencing MUC16expression were detected by Annexin V-FITC/PI, CaspACE Assay System, Colorimetric and Transwell assay.Results1. The results of western blot show:①PV16E6is not expressed in C33A cells, expressed in CaSki cells and SiHa cells.②the expression of MUC16in C-33A cells was significantly lower than CaSki cells and SiHa cells (P<0.01), whereas there was no significant difference in expression of MUC16in CaSki cells and SiHa cells(P>0.05).2. The results of immunohistochemical show:The expression of MUC16in HPV16E6positive normal cervical squamous epithelium, CIN and cervical squamous carcinoma tissues were significantly higher than that of HPV16E6negative normal cervical squamous epithelium, CIN and cervical squamous carcinoma tissues (the correlation coefficient were0.531,0.629and0.452, P<0.01).3. The identification of the eukaryotic expression recombinanted plasmids of MUC16shRNAThe results showed that:gene coding sequences of shRNA are exactly the same and the directions are correct. 4. The shRNA of targeting MUC16mRNA silencing MUC16expression efficiency on CaSki cells.The results of RT-PCR show:compared with CaSki cells and CaSki-shRNA-vect cells, the expressions of MUC16mRNA was reduced obviously in CaSki-shRNA-MUC16cells, and silencing efficiency was93.137%(P<0.01). Whereas there was no significant difference in expression of MUC16mRNA in CaSki cells and CaSki-shRNA-vect cells(P>0.05).The results of western blot show:compared with CaSki cells and CaSki-shRNA-vect cells, the expressions of MUC16protein was reduced obviously in CaSki-shRNA-MUC16cells, and silencing efficiency was93.913%(P<0.01). Whereas there was no significant difference in expression of MUC16protein in CaSki cells and CaSki-shRNA-vect cells(P>0.05).Taken the above results together, CaSki-shRNA-MUC16cells were selected as the experimental group and CaSki-shRNA-vect cells were selected as the control group for further study.5. The apoptosis, migration and invasion of CaSki cells after silencing MUC16expression:①The results of Annexin V-FITC/PI and Flow cytometry indicated that:the mean of apoptotic cells in CaSki-shRNA-MUC16cells were significantly higher than in CaSki-shRNA-vect cells(P<0.01), the percentage of apoptotic cells were5.598%和0.150%.②he results of CaspACETM Assay System, Colorimetric:the activity of Caspase-3in CaSki-shRNA-MUC16cells were significantly higher than in CaSki-shRNA-vect cells(P<0.01).③The results of Transwell migration and invasion assays demonstrated that:the cells which thoughed the basement membrane in the CaSki-shRNA-MUC16group were less than that in CaSki-shRNA-vect group(P<0.01).Conclusions1. The expression of MUC16was positively correlated with E6expression on HPV16E6positive and negative cervical carcinoma cells lines and cervical tissues. 2. The expression of targeted MUC16silence can promote apoptosis and inhibit the ability of metastasis and invasion in HPV16-related cervical carcinoma CaSki cells.