HPV E6通过Rap1信号通路影响宫颈癌细胞增殖、侵袭及迁移的研究  

Effect of HPV E6 on Proliferation,Invasion and Migration of Cervical Cancer Cells Through Rap1 Signaling Pathway

作　　者：秦祥川 李金秋 黄晓婧 忽吐比丁·库尔班 阿仙姑·哈斯木[1] QIN Xiangchuan;LI Jinqiu;HUANG Xiaojing;HUTUBIDING·Kuerban;AXIANGU·Hasim(Basic Medicine College of Xinjiang Medical University/Xinjiang Key Laboratory of Molecular Biology of Endemic Diseases,Urumqi Xinjiang 836001,China)

机构地区：[1]新疆医科大学基础医学院/新疆地方病分子生物学重点实验室,新疆乌鲁木齐836001

出　　处：《昆明医科大学学报》2024年第9期9-16,共8页Journal of Kunming Medical University

基　　金：国家自然科学基金资助项目(82260516)。

摘　　要：目的探讨人乳头瘤病毒E6蛋白(human papillomavirus E6 protein,HPV E6)对宫颈癌Hela细胞增殖、侵袭和迁移的影响及潜在分子机制。方法基于Illumina Hiseq 4000转录组测序技术检测12例不同程度的宫颈病变组织之间的基因表达特征,筛选与HPV相关的差异基因;利用GO、KEGG Pathway方法对差异基因进行生物学功能富集分析;Western blotting检测正常宫颈上皮细胞HCerEpic、宫颈癌细胞Hela中Rap、Rap1GAP蛋白表达水平;通过平板克隆实验、Transwell实验和划痕实验检测其增殖、侵袭及迁移能力;通过sh-E6慢病毒转染下调细胞内HPV E6表达,分为对照组(Hela细胞)、空载组(sh-NON)和sh-E6组(低表达HPV E6);下调HPV E6表达,采用平板克隆和CCK-8实验分别检测每组细胞增殖能力和细胞活力;Transwell和划痕实验检测细胞侵袭、迁移能力;Western blotting检测各组细胞中E6、Rap1、Rap1GAP、CyclinD1、E-cadherin、N-cadherin蛋白的表达水平。在sh-E6组基础上过表达Rap1,Western blot检测细胞中Rap1通路及相关蛋白的表达水平;平板克隆实验、Transwell实验和划痕实验分别检测细胞增殖、侵袭和迁移能力。结果测序结果显示,与正常宫颈HPV阴性比较,HPV阳性的正常宫颈、CIN、宫颈癌组织中差异上调表达的基因分别有340、864和1036个;3个数据集寻找共有差异基因共24个。GO|KEGG富集分析24个差异表达基因主要富集在Rap1相关信号通路。与HCerEpic细胞相比,Hela细胞内Rap1表达升高,Rap1GAP表达降低(P<0.01);其细胞的增殖、侵袭和迁移能力增强(P<0.01);下调HPV E6表达后,与Hela组比较,sh-E6组细胞的增殖、细胞集落形成、侵袭和迁移能力降低(P<0.001);Western blotting表明,与Hela组比较,sh-E6组细胞内Rap1、CyclinD1、N-cadherin蛋白表达降低,Rap1GAP和E-cadherin蛋白表达升高(P<0.001)。与sh-E6组相比,过表达Rap1组(sh-E6/OE Rap1)细胞内Rap1/Rap1GAP、CyclinD1、E-cadherin和N-cadherin蛋白表达被恢�Objective To investigate the effect of human papillomavirus E6 protein(HPV E6)on the proliferation,invasion and migration of Hela cells of cervical cancer and its potential molecular mechanism.Methods Illumina Hiseq 4000 transcriptome sequencing technology was employed to identify the gene expression profiles of 12 cases featuring various degrees of cervical lesions and to screen for HPV-related differential genes.The biological function enrichment of the differential genes was analyzed using GO and KEGG Pathway.Western blotting was utilized to detect the expression levels of Rap and Rap1GAP proteins in normal cervical epithelial cells HCerEpic and cervical cancer cells Hela.The capabilities of proliferation,invasion,and migration were examined by plate cloning,Transwell,and scratch tests.Intracellular HPV E6 expression was down-regulated through sh-E6 lentivirus transfection and classified into the control group(Hela cells),the no-load group(sh-NON),and the sh-E6 group(with low expression of HPV E6).The expression of HPV E6 was diminished,and the proliferation ability and cell viability of each group were determined by plate cloning and CCK-8 assay.Transwell and scratch assays were adopted to detect cell invasion and migration.Western blotting was conducted to detect the expression levels of E6,Rap1,Rap1GAP,CyclinD1,E-cadherin,and N-cadherin in cells of each group.Rap1 was overexpressed based on the sh-E6 group,and the expression levels of the Rap1 pathway and related proteins were measured by Western blot.Cell proliferation,invasion,and migration were evaluated respectively by plate cloning,Transwell assay,and scratch assay.Results The sequencing results indicated that,in contrast to normal cervical HPVnegative tissues,340,864 and 1036 differentially upregulated genes were respectively present in HPV-positive normal cervix,CIN and cervical cancer tissues.A total of 24 differential genes were identified across the 3 datasets.GO|KEGG enrichment analysis of the 24 differentially expressed genes primarily focused o

关 键 词：HPV E6 宫颈癌 Rap1通路 增殖 侵袭 迁移 

分 类 号：R737.33[医药卫生—肿瘤]