miR-449a/b负反馈调节E2F1抑制胃癌细胞增殖  

miR-449a/b negatively regulates E2F1 to suppress proliferation of gastric cancer cells

作　　者：林爱琴[1] 卜文婕[1] 汪萍[1] 高继光[1] 杨建课[1] 丁飞雨 李铁臣[1] LIN Aiqin;BUWenjie;WANG Ping;GAO Jiguang;YANG Jianke;DING Feiyu;LI Tiechen(Department of Medical Biology,Wannan Medical College,Wuhu 241002,China;Clinical College of Medicine,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院医学生物学教研室,安徽芜湖241002 [2]皖南医学院临床医学院,安徽芜湖241002

出　　处：《南方医科大学学报》2020年第1期13-19,共7页Journal of Southern Medical University

基　　金：安徽省高校自然科学研究重点项目(KJ2017A252、KJ2019A0426);安徽省重点实验室自主研究课题(LAB201605);皖南医学院校中青年课题(WK201904)

摘　　要：目的探讨miR-449a/b在胃癌发生中的可能作用机制。方法采用qRT-PCR方法检测了miR-449a/b和E2F1基因在胃癌细胞BGC-823和胃粘膜细胞GES-1表达情况;采用瞬时转染技术将miR-449a/b模拟物(mimic)以及mimic阴性对照分别转入胃癌细胞BGC-823中,qRT-PCR验证转染成功后,通过CCK-8法检测胃癌细胞的增殖能力,流式细胞术检测胃癌细胞凋亡的变化,细胞划痕实验检测胃癌细胞的迁移能力,Western blot方法检测miR-449a/b上调后其靶基因蛋白的表达水平;并通过转染E2F1过表达质粒和空质粒对照入胃癌细胞BGC-823,Western blot确定转染效率后,分别用qRT-PCR和数字PCR检测miR-449a/b的表达水平。结果相比于正常胃粘膜细胞株GES-1,miR-449a/b在胃癌细胞株BGC-823中表达均下调(P<0.01);过表达miR-449a/b可抑制胃癌细胞BGC-823增殖和迁移,并促进其凋亡(P<0.05);过表达的E2F1可上调miR-449a/b的表达(P<0.001),而上调miR-449a/b的表达,其直接靶基因CDK4、CDK6表达下调,并可通过CDKs-pRb-E2F1信号通路,间接下调E2F1蛋白的表达(P<0.01)。结论 miR-449a/b的低表达和E2F1的高表达均参与了胃癌的发生、发展,并且miR-449a/b能负反馈调节E2F1抑制胃癌细胞增殖。Objective To investigate the possible role of miR-449 a/b in the occurrence of gastric cancer. Methods The expression of miR-449 a/b and E2 F1 mRNA in gastric cancer cells BGC-823 and gastric mucosal cells GES-1 were detected with qRT-PCR.miR-449 a/b mimics or a negative control was transiently transfected into BGC-823 cells, and the changes in cell proliferation,apoptosis, and migration ability were assessed using CCK-8 assay, flow cytometry, and scratch wound healing assay,respectively. Western blotting was used to observe the effects of miR-449 a/b upregulation on its target gene expression. The effects of transfection with an E2 F1-over-expressing plasmid or an empty plasmid were analyzed on the expression level of miR-449 a/b in BGC-823 cells using qRT-PCR and digital PCR. Results The gastric cancer cell line BGC-823 showed significantly a lowered expression of miR-449 a/b compared with the normal gastric mucosal cell line GES-1(P<0.01).Overexpression of miR-449 a/b obviously inhibited the proliferation and migration and promoted apoptosis of BGC-823 cells(P<0.05). Overexpression E2 F1 in the cells resulted in significantly up-regulated expression of miR-449 a/b(P<0.001). Upregulation of miR-449 a/b caused significant down-regulation of its direct target genes CDK4 and CDK6 and also of the expression of E2 F1 protein via the CDKs-pRb-E2 F1 signaling pathway. Conclusion The low expression of miR-449 a/b and the high expression of E2 F1 are both involved in the occurrence and progression of gastric cancer, and miR-449 a/b negatively regulates E2 F1 to inhibit the proliferation of gastric cancer cells.

关 键 词：胃癌 miR-449a/b E2F1 细胞增殖 

分 类 号：R735.2[医药卫生—肿瘤]