Effects Of HPV18-E6 Gene SiRNA On Cultured Hep-2 Cells

Objective:The huma n papilloma virus(HPV)E6 gene is one of it's mostimportant cancer gene . Here we constructed the expression vectors siRNA, thattargeted to the oncogene HPV18-E6 gene , respectively. The constructs wereintroduced into the huma n laryngeal carcinoma cancer cells line Hep-2 cellswhich express HPV18-E6 gene, The transfected cells exhibited a much lower rateof growth and proliferation in comparison with the untransfected cells and cellsmock transfected.Methods:(1)The survivin gene promoter region was amplified bypolymerase chain reaction and cloned into pSilencer-4 .1 vector to generate twoplasmid. The HPV18-E6 gene siRNA eukaryotic expression vector wasconstructed by inserting siRNA cDNA into pSilencer 4.1-svv vector siRNAinsertion position.(2)HPV18-E6 gene was amplified by polymerase chainreaction and cloned into pGEM-T Easy vector. This vector were digested andright size bands were gel received.Then we use this plasmid to construct thestanded plasmid for the real-ime PCR in next step(. 3)The HPV18-E6 siRNA wastransfected into Hep-2 cells line by lipofectamine 2000. Sellected the cell line forthree weeks using G418.(4)The expression level of HPV18E6 mRNA weredetected by using real-time quantitative reverse transcription polymerase chain reaction ( real-time RT-PCR). The expression of HPV18-E6 sRNA on Hep-2cells was observed by Western blot .(5)The effect of HPV18-E6 siRNA onHep-2 cell cycle was determined by flow cytometry .MTT were used to measurethe cell grow.Results:(1)Enzyme digestion analysis and DNA sequencing showed tha twe successfully designed expression vector of sRNA targeting specifica lly toHPV18-E6 mRNA, named: pSilencer4.1-Surp neo-E1;Silencer4.1- Surp neo-E2.(2)Enzyme digestion analysis and DNA sequencing showed that we successfullygained the target HPV18-E6 gene by polymerase chain reaction and is cloned intopGEM-T Easy vector .In the same way,we construct the standed plasmid of thereal-ime PCR.(3)The growth of Hep-2 cells was significa ntly suppressed byHPV18-E6 siRNA. The FCM shows the number of cells in G1 phase wasincreased after HPV18-E6 sRNA transfection. The cell division was blocked inG1 pre-DNA-synthetic gap.Conclusion: The present study provides in vitro, these two plasmids had agood contribution in down regular the HPV18-E6 gene expression in Hep-2 cells.The transfected cells exhibited a much lower rate of growth and proliferation incomparison with the untransfected cells and cells mock transfected with apSilemcer vector. The HPV18-E6 gene expression ma y pla y a important role inthe growth of laryngea l carcinoma cells, it might be a new target of the treatmentfor laryngeal carcinoma .