miR-126过表达和ADAM9基因沉默对胃癌SGC-7901细胞生物学行为的影响及其机制  

Effects of miR-126 over-expression and ADAM9 gene silencing on biological behavior of gastric cancer SGC-7901 cells and their mechanisms

作　　者：魏海峰[1] 倪志强[1] 魏雁虹[1] 王起来[1] 李首庆[1] 马寅芙[1] 谭岩[1] 方艳秋[1] WEI Haifeng;NI Zhiqiang;WEI Yanhong;WANG Qilai;LI Shouqing;MA Yinfu;TAN Yan;FANG Yanqiu(Key Laboratory of Biotherapy and Gene Diagnosis,People’s Hospital,Jilin Province,Changchun 130021,China)

机构地区：[1]吉林省人民医院生物治疗与基因诊断重点实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2024年第2期310-319,共10页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然科学基金项目(20200201528JC,20200201572JC);吉林省卫健委卫生健康科技能力提升项目(2021JC056,2021JC057)。

摘　　要：目的:探讨微小RNA-126(miR-126)过表达和解整联蛋白金属蛋白酶9(ADAM9)基因沉默对胃癌细胞生物学行为的影响,并阐明其作用机制。方法:体外培养人胃低分化腺癌SGC-7901细胞和人正常胃黏膜上皮NGEC细胞,提取细胞中总RNA,采用实时荧光定量PCR(RT-qPCR)法检测2种细胞中miR-126和ADAM9 mRNA表达水平。将处于对数生长期的SGC-7901细胞分为miR-126过表达组(miR-126-OE组)和ADAM9基因沉默组(ADAM9 siRNA组),以LipofectamineTM 2000为载体分别转染miR-126模拟物(miR-126 mimics)和敲低ADAM9的RNA寡核苷酸,并设置相应的阴性对照组。采用噻唑蓝(MTT)法检测各组细胞增殖活性,细胞划痕实验检测各组细胞迁移率,Transwell小室实验检测各组细胞的迁移细胞数和侵袭细胞数,Western blotting法检测各组细胞中E-钙黏蛋白、N-钙黏蛋白和波形蛋白表达水平。TargerScan网站预测miR-126的靶基因并通过双荧光素酶报告基因实验验证miR-126和ADAM9的靶向调控关系,采用RT-qPCR法和Western blotting法检测转染miR-126 mimics后SGC-7901细胞中ADAM9 mRNA和蛋白表达水平。结果:RT-qPCR法检测,与人正常胃黏膜上皮NGEC细胞比较,胃癌SGC-7901细胞中miR-126表达水平明显降低(P<0.05),而ADAM9mRNA表达水平明显升高(P<0.05)。MTT法检测,SGC-7901细胞过表达miR-126或沉默ADAM9基因48和72 h后,与相应阴性对照组比较,miR-126 OE组和ADAM9 siRNA组细胞增殖活性均明显降低(P<0.05或P<0.01)。细胞划痕实验检测,与相应阴性对照组比较,48 h后miR-126 OE组和ADAM9 siRNA组细胞迁移率明显降低(P<0.05)。Transwell小室实验检测,与相应阴性对照组比较,miR-126 OE组和ADAM9 siRNA组迁移细胞数和侵袭细胞数明显减少(P<0.05或P<0.01)。Western blotting法检测,与相应阴性对照组比较,miR-126-OE组和ADAM9 siRNA组细胞中E-钙黏蛋白表达水平明显升高(P<0.05或P<0.01),而N-钙黏蛋白和波形蛋白表达水平明显降低(P<0.05或P<0.01)。靶基因Objective:To discuss the effects of microRNA-126(miR-126)over-expression and disintegrin and metalloproteinase 9(ADAM9)gene silencing on the biological behavior of the gastric cancer cells,and to clarify the mechanism.Methods:The human poorly differentiated gastric adenocarcinoma SGC-7901 cells and human normal gastric mucosa epithelial NGEC cells were cultured in vitro.The total RNA was extracted from the cells,and the expression levels of miR-126 and ADAM9 mRNA in both types of cells were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.The SGC-7901 cells at logarithmic growth phase were divided into miR-126 over-expression group(miR-126-OE group)and ADAM9 gene silencing group(ADAM9 siRNA group).The transfection with miR-126 mimics(miR-126)mimics and ADAM9 RNA oligonucleotides were conducted by LipofectamineTM 2000,and the corresponding negative control group was established;MTT assay was used to detect the proliferation activities of the cells in various groups;cell wound assay was used to detect the migration rate of the cells in various groups;Transwell chamber assay was used to detect the the numbers of migration and invasion cells in various groups;Western blotting method was used to detect the expression levels of E-cadherin,N-cadherin,and vimentin proteins in the cells in various gorups.The miR-126 target genes were predicted by TargetScan website,and the targeting regulatory relationship between miR-126 and ADAM9 was confirmed by dual-luciferase reporter assay.The expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics were detected by RT-qPCR and Western blotting methods.Results:The RT-qPCR results showed that compared with human normal gastric mucosa epithelial NGEC cells,the expression level of miR-126 in the gastric cancer SGC-7901 cells was significantly decreased(P<0.05),while the expression level of ADAM9 mRNA was significantly increased(P<0.05).The MTT assay results showed that after 48 and 72 h of over-expressing miR-126 or silen

关 键 词：胃肿瘤 微小RNA-126 靶基因 解整联蛋白金属蛋白酶9 上皮-间质转化 

分 类 号：R735.2[医药卫生—肿瘤]