miR-34a-5p对胃腺癌细胞凋亡的影响及其作用机制

摘要

目的 探讨miR-34a-5p对胃腺癌细胞凋亡的影响及其作用机制.方法 ①采用生物信息学技术预测Bcl-2是否为miR-34a-5p的靶基因.②体外培养人正常胃黏膜上皮细胞RGM-1、人胃腺癌细胞SGC7901,采用qRT-PCR法检测两种细胞miR-34a-5p、Bcl-2 mRNA表达.③将SGC7901细胞随机分为观察组和对照组,观察组转染miR-34a-5p mimic质粒,对照组转染scramble质粒.转染48 h,采用qRT-PCR法、Western blotting法检测Bcl-2 mRNA和蛋白表达.④将SGC7901细胞随机分为阴性对照组、Bcl-2 WT组、Bcl-2 MT组,阴性对照组转染miR-34a-5p mimic和pRL-TK,Bcl-2 WT组转染miR-34a-5p mimic、Bcl-2野生型载体和pRL-TK,Bcl-2 MT组转染miR-34a-5p mimic、Bcl-2突变型载体和pRL-TK.转染48h,采用双荧光素酶报告基因实验检测各组相对荧光素酶活性.⑤将SGC7901细胞随机分为阴性对照组、miR-34a-5p mimic组、pcDNA3.1-Bcl-2组、miR-34a-5p mimic+ pcDNA3.1-Bcl-2组,阴性对照组转染scramble+ pcDNA3.1-空载体,miR-34a-5p mimic组转染miR-34a-5p mimic,pcDNA3.1-Bcl-2组转染pcDNA3.1-Bcl-2,miR-34a-5p mimic+ pcDNA3.1-Bcl-2组转染miR-34a-5p mimic+ pcDNA3.1-Bcl-2.转染48 h,采用流式细胞仪检测各组细胞凋亡率.结果 ①Bcl-2为miR-34a-5p的靶基因.②SGC7901细胞miR-34a-5p mRNA相对表达量低于RGM-1细胞(P＜0.01),Bcl-2 mRNA相对表达量高于RGM-1细胞(P＜0.01).③观察组Bcl-2 mRNA和蛋白相对表达量均低于对照组(P均＜0.01).④Bcl-2 WT组相对荧光素酶活性低于阴性对照组和Bcl-2 MT组(P均＜0.01).⑤miR-34a-5p mimic组细胞凋亡率高于阴性对照组,pcDNA3.1-Bcl-2组、miR-34a-5p mimic+ pcDNA3.1-Bcl-2组低于阴性对照组及miR-34a-5p mimic组,组间比较P均＜0.01.结论 miR-34a-5p可通过靶向调控Bcl-2抑制胃腺癌细胞凋亡,进而参与胃腺癌的发生、发展.%Objective To investigate the effect and mechanism of miR-34a-5p on the apoptosis of gastric adenocarcinoma.-Methods ①Bioinformatics tools were used to predict the potential target gene of miR-34a-5p.②Normal gastric mucosa epithelial cells RGM-1 and gastric adenocarcinoma cells SGC7901 were cultured,and qRT-PCR was performed to detect the expression of miR-34a-5p and Bcl-2 mRNA in these two cell lines.③SGC7901 cells were assigned into two groups:the observation group was transfected with miR-34a-5p mimic plasmid and the control group with scramble plamid for 48 h,and then qRT-PCR was performed to detect Bcl-2 mRNA,and Western blotting was used to detect the Bcl-2 protein.④SGC7901 cells were assigned into three groups:the negative control group was co-transfected with miR-34a-5p mimic and pRL-TK;the Bcl-2 WT group was co-transfected with miR-34a-5p mimic,Bcl-2 WT plasmid,and pRL-TK;the Bcl-2 MT group was co-transfected with miR-34a-5p mimic,Bcl-2 MT plasmid,and pRL-TK.The relative luciferase activity was measured by dual luciferase reporter assay at 48 h.⑤SGC7901 cells were assigned into four groups:the negative control group was co-transfected with miRNA scramble and pcDNA3.1 empty vector,the miR-34a-5p mimic group was transfected with miR-34a-5p mimic,the pcDNA3.1-Bcl-2 group was transfected with pcDNA3.1-Bcl-2,and the miR-34a-5p mimic + pcDNA3.1-Bcl-2 group was co-transfected with miR-34a-5p mimic and pcDNA3.1-Bcl-2.The apoptotic rate of the four groups was measured by flow cytometry after 48 h of transfection.Results ①Bcl-2 was predicted to be the potential target gene of miR-34a-5p.② Compared with RGM-1 cells,SGC7901 cells had relatively low expression of miR-34a-5p and high expression of Bcl-2 mRNA (both P ＜ 0.01).③ The relative expression of Bcl-2 mRNA and protein in the observation group was lower than that in the control group (P ＜ 0.01).④The relative luciferase activity of the Bcl-2 WT group was lower than that of the negative control group and Bcl-2 MT group (P ＜ 0.01).⑤The apoptotic rate of the miR-34a-5p mimic group was higher than that of the negative control group,and the apoptotic rates of the pcDNA3.1-Bcl-2 group and miR-34a-5p mimic + pcDNA3.1-Bcl-2 group were lower than those of the negative control group and miR-34a-5p mimic (P ＜ 0.01).Conclusion The miR-34a-5p can inhibit the apoptosis of gastric adenocarcinoma by up-regulating the expression of Bcl-2 and thus be involved in the occurrence and development of gastric adenocarcinoma.