miR-98-5p靶向调控AKT2基因对人胃癌MGC803细胞增殖的影响

doi:10.6040/j.issn.1671-7554.0.2015.1266

山东大学学报（医学版） ›› 2016, Vol. 54 ›› Issue (11): 27-31.doi: 10.6040/j.issn.1671-7554.0.2015.1266

miR-98-5p靶向调控AKT2基因对人胃癌MGC803细胞增殖的影响

李帅,李迪诺,袁超凡,梁旭,王玉彬

锦州医科大学附属第一医院普外科, 辽宁锦州 121001

收稿日期:2015-12-11 出版日期:2016-11-10 发布日期:2016-11-10
通讯作者: 王玉彬. liaoyilishuai@163.com E-mail:iaoyilishuai@163.com

Inhibitory effect of miR-98-5p on human gastric cancer MGC803 cell proliferation by regulating AKT2 expression

LI Shuai, LI Dinuo, YUAN Chaofan, LIANG Xu, WANG Yubin

Department of General Surgery, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121001, Liaoning, China

Received:2015-12-11 Online:2016-11-10 Published:2016-11-10

摘要/Abstract

摘要： 目的探讨人胃癌MGC803细胞中miR-98-5p和AKT2基因的表达,阐明miR-98-5p对AKT2基因的靶向调控作用以及对MGC803细胞增殖的影响。方法培养MGC803细胞并应用免疫组织化学技术检测AKT2基因的表达;采用生物信息学方法对miR-98-5p和AKT2基因的匹配关系进行预测并用双荧光素酶报告基因系统鉴定;转染miR-98-5p模拟物后,行real-time PCR检测miR-98-5p和AKT2基因的表达,Western blotting检测AKT2蛋白的表达,MTT法和平板克隆形成实验检测癌细胞的增殖情况。结果倒置相差显微镜下可见人胃癌MGC803细胞呈梭形或多角形,少数呈类圆形;免疫组织化学染色显示胞质内有AKT2蛋白的表达,呈棕褐色。靶基因预测软件miRanda和TargetScan显示miR-98-5p和AKT2基因匹配良好,双荧光素酶报告基因系统鉴定发现miR-98-5p能够结合AKT2 mRNA 3'UTR并有效抑制其表达。Real-time PCR和Western blotting检测结果均表明miR-98-5p的过表达能够下调AKT2基因表达。MTT法和平板克隆形成实验表明,miR-98-5p的过表达能够抑制MGC803细胞增殖。结论 miR-98-5p通过靶向作用于AKT2 mRNA 3’UTR负性调控人胃癌MGC803细胞中AKT2的表达,并抑制癌细胞的增殖。

关键词: 人胃癌MGC803细胞, 微小RNA, 丝氨酸/苏氨酸蛋白激酶2

Abstract: Objective To investigate the expressions of miR-98-5p and AKT2 in human gastric cancer MGC803 cells, in order to explore the regulatory effect of miR-98-5p on AKT2 and on the proliferation of MGC803 cells. Methods MGC803 cells were cultured in vitro and cellular morphology changes were observed with phase contrast microscope. The expression of AKT2 in MGC803 cells was determined with immunocytochemistry. The regulatory relation of miR-98-5p and AKT2 was predicted by bioinformatics and identified using dual luciferase report system. Expressions of miR-98-5p and AKT2 were determined using real-time PCR and Western blotting after transfection of miR-98-5p mimics. The proliferation of MGC803 cells was detected by MTT assay and plate colony formation assay. Results Under phase contrast microscope, most of the MGC803 cells were fusiform or polygonal, few were quasi-circular. Immunocytochemistry indicated AKT2 expression in the cytoplasm, which looked sepia. MiRanda and TargetScan showed that miR-98-5p was well matched with AKT2 gene. Dual luciferase reporter system indicated that miR-98-5p could inhibit the expression of AKT2 by binding to its mRNA 3'UTR. Real-time PCR and Western blotting showed that over-expression of miR-98-5p down-regulated expression of AKT2 and suppressed the proliferation of MGC803 cells. Conclusion The miR-98-5p may negatively regulate the expression of AKT2 in human gastric cancer MGC803 cells and inhibit cell proliferation.

Key words: Serine-threonine kinase 2, miRNA, Human gastric cancer MGC803 cells

中图分类号:

R735.2

李帅,李迪诺,袁超凡,梁旭,王玉彬. miR-98-5p靶向调控AKT2基因对人胃癌MGC803细胞增殖的影响[J]. 山东大学学报（医学版）, 2016, 54(11): 27-31.

LI Shuai, LI Dinuo, YUAN Chaofan, LIANG Xu, WANG Yubin. Inhibitory effect of miR-98-5p on human gastric cancer MGC803 cell proliferation by regulating AKT2 expression[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2016, 54(11): 27-31.

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miR-98-5p靶向调控AKT2基因对人胃癌MGC803细胞增殖的影响

Inhibitory effect of miR-98-5p on human gastric cancer MGC803 cell proliferation by regulating AKT2 expression

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