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)。靶基因预测,ADAM9的3´-UTR含有与miR-126-3p互补的核苷酸序列。双荧光素酶报告基因实验,ADAM9是miR-126靶向负调控的下游基因。SGC-7901细胞转染miR-126 mimics 48 h后,与mimics NC组比较,miR-126 OE组细胞中ADAM9 mRNA和蛋白表达水平均明显降低(P<0.05或P<0.01)。结论:胃癌SGC-7901细胞中miR-126低表达而ADAM9基因高表达。miR-126过表达可抑制胃癌SGC-7901细胞增殖活性、迁移和侵袭能力,其机制可能与miR-126靶向负调控ADAM9并抑制胃癌细胞的上皮-间质转化(EMT)进程有关。
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 silencing the ADAM9 gene in the SGC-7901 cells,compared with the corresponding negative control group,the proliferation activities of the cells in both miR-126-OE and ADAM9 siRNA groups were significantly decreased(P<0.05 or P<0.01).The cell wound assay results indicated that compared with the corresponding negative control group,the migration rates of the cells in both miR-126 OE and ADAM9 siRNA groups 48 h after transfection were significantly decreased(P<0.05).The Transwell chamber assay results showed that the numbers of migration and invasion cells in both miR-126-OE and ADAM9 siRNA groups were significantly lower than those in corresponding negative control group(P<0.05 or P<0.01).The Western blotting method results showed that compared with the corresponding negative control groups,the expression level of E-cadherin protein in the cells in miR-126-OE and ADAM9 siRNA groups were significantly increased(P<0.05 or P<0.01),while the expression levels of N-cadherin and vimentin proteins were significantly decreased(P<0.05 or P<0.01).The target prediction results showed that the 3´-UTR of ADAM9 contains nucleotide sequences complementary to miR-126-3p.The dual-luciferase reporter assay results showed that ADAM9 was a downstream target gene negatively regulated by miR-126.Compared with mimics NC group,the expression levels of ADAM9 mRNA and protein in the SGC-7901 cells after transfected with miR-126 mimics for 48 h were decreased(P<0.05 or P<0.01).Conclusion:The gastric cancer SGC-7901 cells are characterized by low expression of miR-126 and high expression of ADAM9 gene.Over-expression of miR-126 can inhibit the proliferative activity,migration,and invasion capabilities of the gastric cancer SGC-7901 cells;the mechanism may be related to the negative regulation of ADAM9 by miR-126 and the inhibition of epithelial-mesenchymal transition(EMT)process in the gastric cancer cells.
作者
魏海峰
倪志强
魏雁虹
王起来
李首庆
马寅芙
谭岩
方艳秋
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)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
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)。
