Construction Of Lentiviral Vectors Expressing High-risk HPV Oncogenes And Transfection Of Human Oral Epithelial Cells With The Vectors

Objectives Human papillomavirus is a small non-enveloped, double-stranded circular DNA virus. HPV is closely related to cervical cancer, and can be detected in 95% of patients with cervical cancer HPV. A large amount of epidemiological survey show that high-risk HPV is also closely related to the incidence of oral cancer. Current research of the relationship between HPV and oral cancer has focused on the study of high-risk HPV E6 and E7 genes, while ignoring the study of oncogene E5. E5 gene exists in the genome of high-risk HPV, but low-risk HPV which causes benign tumors of the oral cavity generally lacks E5 gene, or lacks the translation initiation codon of E5.Meanwhile previous studies found that E5 gene played an important role in the early development of tumour and even enhanced the malignant transformation of E6 and E7 genes on cells. In this study, three lentiviral vectors expressing HPV16 E5, E6 or E7 oncogene were constructed and successfully transfected human oral epithelial cells, and E5 gene related effects on oral epithelial cells conducted a preliminary analysis, which provided the basis for the study of high-risk HPV E5 gene in the pathogenesis of oral squamous cell carcinoma.Methods(1) HPV16 E5, E6 or E7 oncogene was amplified by PCR and ligated into the lentiviral vector p LVX-Ac GFP-N1 respectively by Bam H I and Eco R I double digestion site to construct lentiviral vectors p LVX-Ac GFP-E5, p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7, then respectively cotransfected 293 T cells with packaging plasmids by liposome after digestion and sequencing verified, viral supernatant was collected to concentrate after 48 hours. The virus titer was determined by stepwise dilution method,then oral epithelial cells respectively transfected with each virus. After puromycin screening,oral epithelial cells with HPV 16 E5, E6, or E7 oncogene stable transfection were constructed, then reverse transcription PCR and western blot assays were performed for verifying HPV16 E5, E6 or E7 expression.(2) Oral epithelial cells stably transfected with HPV 16 E5, E6, or E7 oncogene were divided into group A, B, C and D.Group A: oral epithelial cells stably infected with lentivirus p LVX-Ac GFP-E5,p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7, group B: oral epithelial cells stably infected with lentivirus p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7, group C: oral epithelial cells stably infected with lentivirus p LVX-Ac GFP-E5, group D: oral epithelial cells stably infected with lentivirus p LVX-Ac GFP-N1, group E: untransfected oral epithelial cells as blank control group. Preliminary analysis of high-risk HPV E5 gene in the pathogenesis of oral squamous cell carcinoma was carried out by comparing cell adhesion rate,cell proliferation and the expression of E6, E7 m RNA among groups.Results(1) E5, E6 and E7 genes were successfully obtained, double digestion and DNA sequencing showed that insertion sequence of the recombinant lentiviral vector was completely correct.(2) Three kinds of lentivirus supernatant were acquired, by stepwise dilution method the virus titer of lentivirus vectors p LVX-Ac GFP-E5,p LVX-Ac GFP-E6 and p LVX-Ac GFP-E7 were 3 × 108 TU / ml, 2 × 108 TU / ml and 2 × 108 TU / ml respectively.(3) Oral epithelial cells stably expressing HPV 16E5, E6 or E7 oncogene were acquired after puromycin screening, HPV16 E5, E6 or E7 expression was confirmed in oral epithelial cells through reverse transcription PCR and western blot assays, namely, HPV16 E5, E6 or E7 oncogene could successfully express in oral epithelial cells through lentivirus vector.(4) It was found that oral epithelial cells transfected with E5 could not promote cell adhesion and proliferation by comparing and analyzing cell adhesion rate and proliferation among groups(P>0.05), but it did when co-transfected with E5, E6 and E7 genes(P <0.05); It was also found that oral epithelial cells co-transfected with E5, E6 and E7 genes could upregulate the expression of E6 and E7 m RNA comparing with oral epithelial cells co-transfected with E6 and E7 by Real time-PCR(P <0.05).Conclusions(1) Three lentiviral vectors expressing HPV16 E5, E6 or E7 oncogene can successfully transfect human oral epithelial cells.(2) The single high-risk HPV E5 gene transfecting human oral epithelial cell can not promote cell adhesion and proliferation, but it does when high-risk HPV E6 and E7 genes co-transfecting, and the high-risk HPV E5 gene can indirectly upregulate the expression of E6 and E7 m RNA,which provides the foundation for the elucidation of high-risk HPV E5 gene and oral squamous cell carcinoma.