【作者】 李双；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 目的：HPV18 E6癌蛋白与宫颈癌发生、发展密切相关。应用酵母双杂交系统，构建HPV18E6癌蛋白诱饵质粒，在Hela细胞cDNA文库中筛选与HPV18 E6癌基因直接相互结合蛋白，并对TMEM 87B进行生物信息学分析，探讨HPV18 E6和vimentin直接相互作用在顺铂诱导宫颈癌细胞衰老中的功能效应。方法：1．应用RT PCR扩增HPV18E6基因cDNA开放阅读框基因片段后与pGBKT7载体定向重组，通过酶切测序鉴定重组质粒；醋酸锂法将pGBKT7-HPV18 E6重组质粒转化AH109酵母菌株，缺陷性培养基上观察AH109生长情况，检测诱饵载体有无毒性作用和单倍体及二倍体自激活效应。2．将成功构建的pGBKT7-HPV18E6重组质粒醋酸锂法转化感受态酵母菌AH109，随后转化Hela MATCHMAKER cDNA文库质粒，筛选阳性克隆。提取酵母质粒纯化后电转感受态大肠杆菌DH5α，提取阳性质粒，排除假阳性和重复插入片段后酶切电泳、测序后行基因序列分析宫颈癌HPV18 E6直接相互作用蛋白。3．应用因特网资源，运用BLAST、ProtParam tool、The ELM Server及InterProScan等数据库或在线软件对TMEM 87B基因及其编码蛋白进行生物信息学分析，预测其基因结构、染色体定位、编码蛋白质理化性质、亚细胞定位、蛋白质功能域等信息，并对多物种中的相似性蛋白进行了系统进化分析。4．酵母体内和体外Co-IP法验证HPV18 E6和vimentin直接相互作用；免疫组化法检测HPV18 E6和vimentin蛋白及CBX3蛋白在人宫颈癌切片中的表达情况。应用不同浓度梯度顺铂作用Hela细胞，选择最佳诱导衰老而无明显凋亡发生的实验浓度。检测Hela细胞衰老过程中HPV18 E6和vimentin以及P53、P21、CDC2等衰老相关调控基因表达变化；应用SA-βG al(衰老相关β-半乳糖苷酶)染色法检测通过脂质体法单独转染或共转染siRNA-HPV18 E6和siRNA-vimentin时顺铂诱导肿瘤细胞衰老敏感性变化情况；PI单染流式细胞仪检测细胞周期变化；Western blot检测P53、P21、CDC2等衰老相关调控基因表达变化情况；建立顺铂诱导宫颈癌肿瘤细胞衰老动物模型；分析衰老动物模型中HPV18 E6和vimentin表达变化。结果：1．成功构建重组质粒pGBKT7-HPV18 E6，转化重组质粒pGBKT7-HPV18 E6和pGBKT7空载体的酵母菌在YPDA液体培养基中培养16h后，菌液的A600nm值分别为0．98和0．99；两种酵母菌均在SD／-Trp／X-α-gal平板上长出白色菌落，在SD／-His／-Trp／X-α-gal，SD／-Ade／-Trp／X-α-gal平板上不能生长，pGBKT7-HPV18E6转化酵母菌AH109与pGADT7-T转化酵母菌Y187交配后二倍体在SD／-Trp／-Leu培养盘中能长出白色克隆，而在SD／-His／-Trp／-Leu／X-α-gal及SD／-Ade／-His／-Trp／-Leu／X-α-gal盘中均无克隆生长，说明重组质粒pGBKT7-HPV18E6对酵母菌AH109无毒性且不具自主激活报告基因效应。2．该诱饵载体应用于在Hela细胞cDNA文库中筛选与HPV18E6相互作用的蛋白质，随机挑取50个克隆进行电转后，双酶切后电泳，测序鉴定7种与HPV18E6直接相互作用蛋白：跨膜蛋白87B、膦甲酸免疫相关蛋白5、波形蛋白Vimentin、KM-HN-1蛋白、无功能糖基转移酶样蛋白7、痘苗相关激酶蛋白2以及一种未知蛋白。3．对获得的7种阳性蛋白进行初步生物信息学分析确定其生物功能，对TMEM 87B蛋白进行进一步分析，发现TMEM87B是一种6次跨膜的分泌性蛋白，有众多磷酸化位点和功能基序，在多物种间有高度保守序列，与肿瘤生成信号转导和转录调控相关；4．免疫组化结果显示CBX3在CIN和宫颈癌早期表达达高峰，而HPV18 E6和vimentin在宫颈癌早期呈高表达。在一定小剂量顺铂(3．300μM)诱导下，Hela细胞呈现衰老表型，细胞变大变扁平，胞浆空泡增多，SA-βGal染色呈阳性着色，细胞周期阻滞在G2／M期，细胞调亡不明显，P53、P21、P-CDC2基因在顺铂诱导衰老过程中表达增强。分别转染50nM siRNA-vimentin和50nM siRNA-HPV 18 E6后，增敏顺铂(1．650μM)诱导Hela细胞衰老(P＜0．05)；共转染50nM siRNA-vimentin和50nM siRNA-HPV18 E6后，Hela细胞衰老率增加，且增敏顺铂(0．825μM)诱导Hela细胞衰老(P＜0．05)。衰老细胞变大、变园、扁平，衰老相关β-半乳糖苷酶染色阳性；细胞周期G2／M期阻滞；vimentin、P53、CDC-2表达上调，HPV 18 E6表达下调。结论：七种与HPV18E6相互作用的蛋白质可能是潜在的宫颈癌检测指标。TMEM87B是一种6次跨膜的分泌性蛋白，可能与肿瘤生成信号转导和转录调控相关；HPV18 E6和vimentin直接相互作用可能抵抗了顺铂诱导宫颈癌细胞衰老。更多还原

【Abstract】 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.更多还原