Screening And Identification Of Potential Target Genes Of HPV16E6and Their Expressions In Cervical Squamous Cell Carcinoma

Background High risk-human papillomaviruses(HR-HPVs), especially HPV16, have closely relationship with the occurrence and evolution of various malignant tumors such as cervical cancer. Persistent expression of E6and E7oncogene is required for malignant transformation of host cell and malignant phenotype maintenance, of which the E6gene encoding the E6protein plays a role in promoting cell proliferation, invasion and metastasis. Research shows that E6gene has no homology in human genome, and exists only in infected HPVs cells. Therefore, E6provides an ideal target for the treatment of cervical cancer and other HPVs associated tumor. However, E6as the treatment targets are all in the research stage. Although the mechanism HPV16E6induced tumor occurrence and evolution has been reported, but there are differences reported as the mechanism of carcinogenesis is complex.Therefore, in-depth studying the carcinogenic mechanism of HPV16E6is of great significance for exploring new treatment of HPV16associated tumor.Objectives HPV16E6related potential target genes was screened and verified and their expression in HPV16positive and negative cervical squamous carcinoma tissues was observed, both of which were used to lay foundation for studing the regulatory relationship and mechanisms of HPV16E6and the screened differentially expressed genes associated with HPV16E6, enriching the carcinogenic mechanism of HPV16and offerring a new target for the tumor treatment.Methods1. Taken the research group p-Genesil-E6shRNA eukaryotic expression recombinant plasmid of HPV16positive cervical cancer CaSki cells (silencing E6expression)(experimental group) and p-Genesil-1plasmid transfer (including unrelated shRNA) CaSki cells (control group) as the research object, together with Agilent human genome expression profile chip technology, changes in the gene expression profile was detected after silencing HPV16E6expression. Gene expression profile analysis was repeated3times,3results, combined with SBC microarray data analysis system and the GenBank database to screen for differentially expressed genes related to HPV16E6.2. Taken p-Genesil-E6-shRNA-CaSki cells, p-Genesil-unrelated cells, shRNA-CaSki cells, cervical carcinoma SiHa cell (HPV16positive) and C33-A cells (HPV16negative) as research object, HPV16E6related differential expression gene was screened by western blot.3. Taken56cases of collected HPV16E6positive cervical squamous carcinoma tissues and36cases of HPV16E6negative cervical squamous carcinoma tissues as research objects, the expression of HPV16E6related differential expression gene was screened by immunohistochemistry.Results1. Agilent genome expression profile chip analysis results:①The purity and integrity of the RNA sample reached the requirement of chip.②The hybridization effect is ideal.③HPV16E6related significant differential expression genes were screened based on the analysis of three gene expression profile chip results:MUC16gene expression was down-regulated by about5times, MMP3and SLAMF7gene expression was down-regulated by about8times, SERPINA3gene expression was down-regulated by about7times, TRAIL and ANXA10gene expression was up-regulated by about3times, CDH15gene expression was up-regulated by about5times.2.The results of western blot show:①Expression of MUC16and SERPINA3in C33-A cells and p-Genesil-E6-shRNA-CaSki cells was significantly lower than that of SiHa cells and p-Genesil-unrelated shRNA-CaSki cells(P<0.01), whereas there was no significant difference in expression of MUC16and SERPINA3in SiHa cells and p-Genesil-unrelated shRNA-CaSki cells as well as in C33-A cells and p-Genesil-E6-shRNA-CaSki cells (P>0.05).②There was no significant difference in expression of MMP3in C33-A cells, SiHa cells and p-Genesil-unrelated shRNA-CaSki cells (P>0.05), however, expression of MMP3in p-Genesil-E6-shRNA-CaSki cells decreased significantly (P<0.01).③The expression of SLAMF7in p-Genesil-E6-shRNA-CaSki cells was significantly lower than that of C33-A cells, SiHa cells and p-Genesil-unrelated shRNA-CaSki cells(P<0.01), and expression of SLAMF7in C33-A cells was significantly lower than that of SiHa cells and p-Genesil-unrelated shRNA-CaSki cells(P<0.01), but there was no significant difference in expression of SLAMF7in SiHa cells and p-Genesil-unrelated shRNA-CaSki cells(P>0.05).④The expression of TRAIL, ANXA10and CDH15in C33-A cells and p-Genesil-E6-shRNA-CaSki cells was significantly higher than that in SiHa cells and p-Genesil-unrelated shRNA-CaSki cells (P<0.01or0.05), and there was no significant difference in expression of TRAIL, ANXA10and CDH15in SiHa cells and p-Genesil-unrelated shRNA-CaSki cells as well as in C33-A cells and p-Genesil-E6-shRNA-CaSki cells (P>0.05).3.The results of immunohistochemical show:①The expressions of MUC16, MMP3, SLAMF7and SERPINA3in HPV16E6Positive cervical squamous carcinoma tissues were significantly higher than that of HPV16E6negative cervical squamous carcinoma tissues(P<0.01or0.05).②The expressions of TRAIL、ANXA10and CDH15in HPV16E6positive cervical squamous carcinoma tissues were significantly lower than that of HPV16E6negative cervical squamous carcinoma tissues (P<0.01).Conclusions1. MUC16, MMP3, SLAMF7, SERPINA3, ANXA10and CDH15were screened and preliminary proved might to be a potential target gene of HPV16E6.2. TRAIL is a target gene of HPV16E6was further proved.3. It lays foundation for studing the regulatory relationship and mechanisms of HPV16E6and the screened differentially expressed genes associated with HPV16E6.