Construction Of Recombined HPV16 E6-shRNA Lentivirus And Its Suppression To Cervical Cancer Cells

Human papilloma virus (HPV) infection was essential for genesis of cervical cancers. About 50% of cervical cancer was related with HPV type 16 (HPV16). After infection, E6 and E7 gene of HPV were integrated to genome of cervical epithelium. Continued expression of transforming oncoprotein E6 not only drives the neoplastic progression in cervical epithelium but also plays important role in maintaining malignant phenotype of cervical cancer cells. Antisensenucleic acids and targeting ribozyme were usually applied to block E6 gene expression to explore mechanism of cervical cancinogenesis. However, their blocking efficiency was limited, and it was difficult for them to reach cells in vivo. So it is very neccesary to find more efficient blocking method in order to elucidate further transforming mechanism of E6 protein.Therapeutic effect of traditional surgery and radiation was limited in advanced cervical cancer. However, biotherapy gives highlight to it. Since the integrated HPV transforming gene have no appreciable sequence homology to human genome, they become attractive targets for biotherapy. RNA interfering has been utilized to elucidate gene function employing double-strand RNA that is capable of degrading mRNA in a sequence-specific manner. Small hairpin RNA (shRNA), which was transcripted from expression sequence in cells, could silence target gene function over 7 days. Further more, shRNA was very convenient for working in vivo. Lentivirus was characterized with high infection ability and faint stimulation to immune system. Thus, lentivirus would be commendable carrier for shRNA.However, there was not any reference till now about that shRNA carried by lentivirus could block E6 expression. In the hope of developing a gene-specific therapy for HPV related cancer, we designed three expression sequences of shRNA target to HPV16 E6(E6-shRNA), and cloned the sequences to lentivirus work vector PLL3.7. The recombined vectors were identified by restriction enzyme analysis and sequencing. Lentivirus was...