miR-9对人胃癌细胞株活化白细胞黏附分子基因表达的靶向调控作用  

Targeting regulation of miR-9 on the expression of activated leukocyte cell adhesion molecule gene in human gastric cancer cell lines

作　　者：余丹纯[1] 聂玉强[1] 黄耀星[1] 孙小娟[1] YU Danchun;NIE Yuqiang;HUANG Yaoxing;SUN Xiaojuan(Department of Gastroenterology,Guangzhou First People’s Hospital,Guangzhou 510180,China)

机构地区：[1]广州市第一人民医院消化内科,广东广州510180

出　　处：《胃肠病学和肝病学杂志》2019年第1期48-51,共4页Chinese Journal of Gastroenterology and Hepatology

基　　金：2016年广州市卫生和计划生育科技一般引导项目(20161A011001)

摘　　要：目的探讨miR-9对人胃癌细胞株活化白细胞黏附分子(activated leukocyte cell adhesion molecule,ALCAM)基因表达的靶向调控作用。方法运用生物信息学方法预测靶向调控ALCAM的miRNA;应用SYBR Green荧光定量PCR检测各个人胃癌细胞株中ALCAM mRNA的表达情况,确定实验细胞株;通过PCR扩增合成含有miR-9结合位点的ALCAM mRNA 3'端非编码区(ALCAM 3'UTR)片段和在miR-9结合位点进行突变的ALCAM-mut mRNA 3'UTR,测序鉴定后将其克隆至报告基因载体psi CHECK-2质粒,分别命名为psi CHECK-2-ALCAM和psi CHECK-2-ALCAM-mut;分组转染重组质粒和miRNA,双荧光素酶报告基因系统检测各实验组中细胞荧光素酶的表达,SYBR Green荧光定量PCR和Western blotting检测ALCAM mRNA及蛋白表达水平。结果生物信息学分析筛选发现miR-9可能靶向调控ALCAM; ALCAM mRNA在各胃癌细胞株中的表达量明显高于人正常胃黏膜GES-1细胞株,且SGC-7901细胞株ALCAM mRNA的表达量最高(P <0. 05),确定为实验细胞株;测序验证ALCAM 3'UTR和ALCAM-mut3'UTR经PCR扩增合成成功; psi CHECK-2-ALCAM质粒共转染miR-9 mimics细胞组的相对荧光素酶活性较psi CHECK-2-ALCAM质粒共转染miR-NC、miR-9 inhibitor细胞组或空白对照组明显降低(P <0. 05),且该组细胞ALCAM mRNA和蛋白的表达明显降低(P <0. 05);而psi CHECK-2-ALCAM-mut质粒共转染miR-NC、miR-9 mimics或miR-9 inhibitor细胞组的相对荧光素酶活性与空白对照组相比,差异无统计学意义(P> 0. 05)。结论在SGC-7901中,miR-9通过与ALCAM mRNA 3'UTR结合而负性调控ALCAM基因的表达。Objective To investigate the targeting regulation of miR-9 on the expession of activated leukocyte cell adhesion molecule(ALCAM)gene in human gastric cancer cell lines.Methods Bioformatics analysis was used to predict miRNA targeting ALCAM.ALCAM mRNA expression was detected in different cell lines by SYBR Green fluorescence quantitative PCR in order to determine the experimental cell lines.The ALCAM mRNA 3′UTR fragment containing the miR-9 binding site and the ALCAM-mut mRNA 3′UTR mutated at the miR-9 binding site were synthesized by PCR amplification and cloned into the reporter gene vector psiCHECK-2 plasmid.The recombinant plasmids were named psiCHECK-2-ALCAM and psiCHECK-2-ALCAM-mut,respectively.Recombinant plasmids,microRNAs were co-transfected into SGC-7901 cell.Dual luciferase reporter system was used to detect the luciferase expression in each experimental group.SYBR Green fluorescence quantitative PCR and Western blotting were performed to detect the expressions of ALCAM mRNA and protein.Results Bioinformatics analysis and screening found that miR-9 may be targeted regulation of ALCAM.The expression of ALCAM mRNA in gastric cancer cell lines was significantly higher than that in human normal gastric mucosa GES-1 cell lines.SGC-7901 cell line had the highest expression of ALCAM mRNA(P<0.05),which was determined as the experimental cell line.ALCAM 3′UTR and ALCAM-mut 3′UTR were successfully synthesized and confirmed by sequencing.Compared with blank control group,miR-NC group and miR-9 inhibitor group,miR-9 mimics group significantly reduced the relative luciferase activity of cells co-transfected with psiCHECK-2-ALCAM(P<0.05)and the expressions of ALCAM mRNA and protein were significantly decreased(P<0.05).In contrast,the relative luciferase activity of cells co-transfected with psiCHECK-2-ALCAM-mut in miR-9 mimics group,miR-NC group and miR-9 inhibtor group had no significant changes compared with that in blank control group(P>0.05).Conclusion miR-9 negatively regulated the expression of ALCAM ge

关 键 词：胃癌 双荧光素酶报告基因系统 逆转录聚合酶链反应 蛋白质印迹法 MIR-9 活化白细胞黏附分子 

分 类 号：R735.2[医药卫生—肿瘤]