Preliminary Research Of HPV Integration Conditions And E7Gene Silencing In Several Carcinoma Cell Lines

Human papillomavirus (HPV) is a double stranded DNA virus, which can be divided into two main groups-high risk type(HR-HPV) and low risk type(LR-HPV)-according to their capabilities to induce diseases. High risk type HPV will integrate into the host genome when being infected. The integration preferences in human papillomavirus (HPV) have been intensively studied and argued over these years. Very petcuilar and unexpected phenomenons were obtained from this study by employing a nested PCR and quantitative real-time PCR to gain viral-human fusion mRNA sequences and to detect viral copy-numbers in cervical cancer cell lines CaSki, SiHa, and HeLa. In total14fusion mRNAs, and one mRNA sequence contained only viral genes in SiHa were obtained. All the human parts in fusion mRNAs specifically located in8q24.1-24.2, and13q14-31gene desert regions that are related to cancer in each cell line. Interestingly, a39-base overlapped sequence at the human-viral boundary was observed in one mRNA sequences. Moreover, some human parts matched separated segments on human genome, in one case, these corresponding segments on human genome and fusion mRNA sequence were in different orders. In addition, almost all the human parts in fusion mRNAs are triple-copy genes on human chromosomes. Surprisingly, all the viral parts in fusion mRNAs stopped in the first13-18nucleotides in El gene of HPV type18and16except one mRNA contain no human part with as much as85nucleotides of E1. However E6mRNA copies in SiHa is far more complicated than in CaSki.In order to explore the impact of the integrated HPV type18on carcinogenesis of esophageal squamous cancer cell line EC9706. A E7gene silencing sequence was induced into the pSUPER.retro.neo+gfp vector to down regulate the viral mRNA in EC9706cell lines. Real time PCR and western blot was employed to detect the related genes in both mRNA and protein level respectively in these cell lines. Totally two E7gene down regulated stable cell lines was obtained. The cell viability was also detected by the proliferate property. The decrease of E6gene proved the existence of E6-E7mRNA in EC9706cell lines as in these cervical cell lines. The western blot results also show that there was a huge increase in the p27protein which involved in the E7-cellular protein network. The reduced cell viability showed that the expressed viral genes in EC9706cell lines are the factors to enhance the cell viability. This study proved the integrated HPV could be the pathogenetic factor of carcinogenesis in esophageal squamous cancer cell line EC9706, and the promising results established the basis of RNA silencing in therapeutic practice in HPV induced cancer.