miR-34a-5p对胃腺癌细胞凋亡的影响及其作用机制  

Effect of miR-34a-5p on apoptosis of gastric adenocarcinoma

作　　者：朱金峰[1] 曾薇[1] 王海江[1] 

机构地区：[1]新疆医科大学附属肿瘤医院,乌鲁木齐830011

出　　处：《山东医药》2017年第36期21-24,共4页Shandong Medical Journal

基　　金：新疆维吾尔自治区自然科学基金面上项目(2016D01C347)

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48 h,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、pc DNA3.1-Bcl-2组、miR-34a-5p mimic+pc DNA3.1-Bcl-2组,阴性对照组转染scramble+pc DNA3.1-空载体,miR-34a-5p mimic组转染miR-34a-5p mimic,pc DNA3.1-Bcl-2组转染pc DNA3.1-Bcl-2,miR-34a-5p mimic+pc DNA3.1-Bcl-2组转染miR-34a-5p mimic+pc DNA3.1-Bcl-2。转染48 h,采用流式细胞仪检测各组细胞凋亡率。结果 (1)Bcl-2为miR-34a-5p的靶基因。(2)SGC7901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)。(4)Bcl-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)。(5)miR-34a-5p mimic组细胞凋亡率高于阴性对照组,pc DNA3.1-Bcl-2组、miR-34a-5p mimic+pc DNA3.1-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（1）Bioinformatics tools were used to predict the potential target gene of miR-34a-5p.（2）Normal 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.（3） SGC7901 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.（4） SGC7901 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.（5）SGC7901 cells were assigned into four groups： the negative control group was co-transfected with miRNA scramble and pc DNA3. 1 empty vector,the miR-34a-5p mimic group was transfected with miR-34a-5p mimic,the pc DNA3. 1-Bcl-2 group was transfected with pc DNA3. 1-Bcl-2,and the miR-34a-5p mimic ＋ pc DNA3. 1-Bcl-2 group was co-transfected with miR-34a-5p mimic and pc DNA3. 1-Bcl-2. The apoptotic rate of the four groups was measured by flow cytometry after 48 h of transfection. Results（1）Bcl-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）.（4）The relative luciferase activity of the Bcl-2 WT group was lower than that of th

关 键 词：胃癌 微小RNA-34a-5p B细胞淋巴瘤2 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤]