人胃癌细胞Caveolin-1基因的表达与DNA甲基化修饰之间的关系研究  

The Interaction Between Expression and Methylation Status of Caveolin-1 Gene in Human Gastric Cancer Cells

作　　者：毛立祺 戴春艳 金威洋 傅宇斐 陈喆[1] 吕宾[1] 王曦[1] MAO Liqi;DAI Chunyan;JIN Weiyang(Key Laboratory of Digestive Pathophysiology of Zhejiang Province,The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou(310006),China;The First Affilialed Hospital of Hazhou;School of Life Science and Environmental Science,Hangzhou Normal University)

机构地区：[1]浙江中医药大学附属第一医院浙江省消化道疾病病理生理研究重点实验室,杭州310006 [2]湖州市第一人民医院 [3]杭州师范大学生命与环境科学学院

出　　处：《浙江中医药大学学报》2021年第9期939-944,共6页Journal of Zhejiang Chinese Medical University

基　　金：浙江省医药卫生科技计划项目青年人才计划(2019RC228、2019RC229);浙江省自然科学基金项目(LQ14H160014、LY21H030002);国家自然科学基金项目(81503297、81400594、81603340、81773945、81770535)。

摘　　要：[目的]观察胃癌细胞系中小窝蛋白-1(Caveolin-1,Cav-1)基因不同亚型的表达水平,探讨影响其基因转录调控的甲基化修饰途径。[方法]选择Cav-1基因表达水平不同的胃癌细胞系,应用实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测不同胃癌细胞系中Cav-1基因的两个亚型α和β的mRNA水平,之后利用磷酸胞苷酰(基)鸟苷(cytidylyl phosphate guanosine,Cp G)岛在线预测软件对其启动子区和第一外显子区CpG岛分布情况进行分析,最后应用甲基化qPCR对其甲基化水平进行检测。[结果]Real-time q PCR结果显示,人胃癌细胞MGC-803中Cav-1以及Cav-1α的表达分别是人正常胃黏膜上皮细胞GES1的(1.782±0.489)倍和(2.604±0.945)倍,差异均有统计学意义(P<0.05,P<0.01);人胃癌细胞AGS中的Cav-1以及Cav-1α的表达则仅是GES1细胞的(0.017±0.002)倍和(0.0005±0.00003)倍,差异均有统计学意义(P<0.001,P<0.001)。相对于GES1细胞,MGC-803细胞和AGS细胞中Cav-1β的mRNA表达差异无统计学意义(P>0.05)。利用CpG岛在线预测软件分析结果表明,人Cav-1α基因的启动子区存在一个174bp大小的CpG岛,该岛位于相对于转录起始位点(transcription start stecte,TSS)+1的-250~-77区域内,包含了17个CG位点。甲基化qPCR检测发现,Cav-1α基因启动子区-158~-77区域处于去甲基化状态,则Cav-1α的m RNA表达水平较高,相反,该区域处于高度甲基化状态,则其mRNA表达较低。[结论]人胃癌细胞中Cav-1的表达主要与其α亚型的表达相关,与β亚型无关;并且其启动子区CpG岛内-158~-77区域的甲基化修饰程度与其m RNA转录调控水平密切相关。[Objective]To observe the expression of different transcript variants Caveolin-1(Cav-1)gene in human gastric cancer cells,and to investigate the epigenetic mechanism.[Methods]The gastric cancer cell lines expressing different levels of Cav-1 gene were selected and the mRNA levels of two subtypes of Cav-1 gene were detected by Real-time quantitative polymerase chain reaction(Real-time qPCR).Then the status of cytidylyl phosphate guanosine(CpG)island within the promoter region and the first exon region of Cav-1 gene was analyzed by the software of MethPrimer-Design Primers of Methylation PCRs.[Results]Real-time qPCR showed that compared with normal gastric epithelial cells GES1,the expression of Cav-1 and Cav-1αin human gastric cancer cells MGC-803 was(1.782±0.489)folds and(2.604±0.945)folds respectively,the difference was statistically significant(P<0.05,P<0.01);whereas the expression of Cav-1 and Cav-1αin the other human gastric cancer cells AGS was only(0.017±0.002)and(0.0005±0.00003)folds of GES1 cells(P<0.001,P<0.001).However,compared with GES1 cells,there was no significant difference of Cav-1βm RNA expression in MGC-803 and AGS cells(P>0.05).The analysis of CpG island status prediction software showed that there was a Cp G island located between-250~-77 bp relative to+1 within transcription start state(TSS)of Cav-1 gene,which was 174 bp in length and contained 17 CG dinucleotides.The Methyl-qPCR assay showed that the demethylation of the region between-158~-77 of Cav-1 gene was associated with the high level of its m RNA expression.Meanwhile,the methylation of this region was related with the low level.[Conclusion]The expression of Cav-1 in human gastric cancer cells is related to the expression of subtypeαand has no correlation with the subtypeβ,and the methylation of-158~-77 region within the promoter functioned as the major regulation domain of Cav-1 transcription activity.

关 键 词：小窝蛋白-1 亚型 胃癌 甲基化 启动子区 CPG岛 转录调控 基因表达 

分 类 号：R331[医药卫生—人体生理学]