Screening For Binding Proteins Derectly Interacting With Human Papillomavirus Type 18 E6 Oncogene And The Correlated Functional Validation

Subject: High-risk human papillomaviruses oncoprotein 18 E6 (HPV18 E6) is associatedwith cervix cancer. The study was conducted to screen for novel binding proteinsinteracting with high-risk HPV 18 E6 oncogene, to identify Transmembrane Protein 87B(TMEM 87B) as a novel binding protein interacting with HPV 18-E6 oncoprotein andperform an initial bioinformatics analysis, and to explore the effects of the direct interactionbetween HPV 18 E6 and vimentin on the senescence induced by DDP.Methods:1. The strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequenttransference was utilized to screen for interacting proteins with HPV 18 E6 in humanHela cDNA library.2. The yeast strain AH109 was transformed with pGBKT7-HPV 18 E6, and the yeastmating assay was utilized to identify the interaction between TMEM 87B and HPV18E6 in human Hela cDNA library.3. TMEM87B gene structure, genomic localization, the physical and chemicalcharacteristics, subcellular localization, functional domain were predicted, as well as thesystematic evolution analysis on the similar proteins among several species.4. The yeast strain AH109 was transformed with pGBKT7-HPV 18 E6, and the yeastmating assay was utilized to identify the interaction between vimentin and HPV18 E6in human Hela cDNA library. The expression of CBX3, HPV18 E6 and vimentin weredetected in the tissues of cervical infection, CIN and cervical cancer by use of immunohistochemistry. Cisplatin with different concentrations were applied in tumourcells, and to choose the suitable concentration when the senescence rate was highestwithout obvious apoptosis. The cell senescence rates of tumour cells induced bycisplatin were detected by using of SA-βGal staining method.5. The expression and activity of P53, P21 and P-CDC2 genes changed obviously duringthe senescence process. The senescence sensitivity of Hela cells induced by cisplatin(1.650μM) was increase after the transfection of 50nM siRNA-vimentin and 50nMsiRNA-HPV, while the senescence sensitivity of Hela cells induced by cisplatin (0. 825μM)was increase after the co-transfection(P＜0.05). The senescent Hela cells inducedby DDP become large and flatten, positive staining of SA-β-Gal. PI staining methodwas used to test the cell cycle. The expression of senescence regulating genes such asP53, P21 and Cdc2 were detected by using of western blot. The senescence aminalmodel was established and the expression of HPV18 E6 and vimentin was tested.Results:1. In yeast two-hybrid assay, HPV18 E6 mRNA was expressed and there was noself-activation and toxicity in strain AH 109.2. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B,phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein,dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, wereidentified. It was suggested that yeast two-hybrid system is an efficient for screeninginteracting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins,which may be associated with signal transduction and transcriptional control, epithelialcell invasion and migration, as well as humoral and cellular immune etc.3. The special TMEM 87B mRNA expression was detected in Hela cells, and the blueclones were validated in the yeast mating assay. Efficient bioinformatics analysis hasfundamentally identified that TMEM 87B is a secretary protein, containing many phosphorylation sites and functional motifs, and may be involved in signal transductionand transcriptional control in carcinogenesis. It has been indicated that the yeasttwo-hybrid system is an efficient for screening interacting proteins.4. The expression of CBX3 reaches its peak in the tissues of CIN and cervical cancer inthe early stage, while the over-expression of HPV18 E6 and vimentin were detected inthe tissues of cervical cancer in the early stage. The senescent Hela cells induced byDDP (3.300μM) become large and flatten, increased vacuolus in cytoplasm, positivestaining of SA-β-Gal. The senescent cells were mainly blocked during G2/M period andapoptosis was not obvious. The expression and activity of P53, P21 and P-CDC2 geneschanged obviously during the senescence process. The senescence sensitivity of Helacells induced by cisplatin (1.650μM) was increase after the transfection of 50nMsiRNA-vimentin and 50nM siRNA-HPV, while the senescence sensitivity of Hela cellsinduced by cisplatin (0.825μM) was increase after the co-transfection (P＜0.05). Thesenescent Hela cells induced by DDP become large and flatten, positive staining ofSA-β-Gal.Conclusion: This investigation provides functional clues for further exploration of potentialoncogenesis targets for cancer biotherapy. The novel gene TMEM 87B may interact withHPV18 E6, and maybe a potential oncogenesis target according to bioinformatics analysis.The direct interaction between HPV18 E6 and vimentin plays an important role in thesenescence induced by DDP.