miR-143-5p靶向AGR2在肝癌细胞增殖和凋亡中的作用  

Role of miR-143-5p targeting AGR2 in proliferation and apoptosis of hepatocellular carcinoma cells

作　　者：任松[1,2] 杨林青 潘龙飞[3] 田红卫 REN Song;YANG Linqing;PAN Longfei;TIAN Hongwei(Fourth Ward,Department of General Surgery,Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China;National&Local Joint Engineering Research Center of Biodiagnosis and Biotherapy,Second Affiliated Hospital of Xi’an Jiaotong University;Department of Emergency,Second Affiliated Hospital of Xi’an Jiaotong University)

机构地区：[1]西安交通大学第二附属医院普通外科四病区,西安710004 [2]西安交通大学第二附属医院生物诊断治疗国家地方联合工程研究中心 [3]西安交通大学第二附属医院急诊科

出　　处：《山西医科大学学报》2022年第11期1376-1384,共9页Journal of Shanxi Medical University

基　　金：国家自然科学基金青年科学基金项目(81802456);陕西省重点研发计划项目(2020SF-298)。

摘　　要：目的探讨miR-143-5p在肝癌细胞增殖和凋亡中的作用及其机制。方法qRT-PCR检测正常肝细胞HL-7702和肝癌细胞(BEL-7402、HepG2、SMMC-7721、MHCC-97H和Hep3B)中的miR-143-5p表达。选取肝癌细胞BEL-7402和HepG2,将每株肝癌细胞分别分为:mimics-NC组(转染阴性对照mimics-NC)和miR-143-5p mimics组(转染miR-143-5p mimics)。采用MTT法、克隆形成实验、流式细胞术和Western blot分别检测mimics-NC组和miR-143-5p mimics组细胞增殖能力、克隆形成能力、凋亡率和凋亡相关基因(Bax、Caspase-3和Bcl-2)的表达。通过靶基因预测网站RNA22预测miR-143-5p的靶基因。双荧光素酶报告基因验证miR-143-5p和靶基因前梯度蛋白2(AGR2)关系。qRT-PCR和Western blot检测mimics-NC组和miR-143-5p mimics组肝癌细胞BEL-7402和HepG2中AGR2的表达水平。再将肝癌细胞BEL-7402和HepG2分别分为:si-NC组和si-AGR2组,分别转染阴性对照si-NC和si-AGR2。采用MTT、克隆形成实验和流式细胞术分别检测si-NC组和si-AGR2组细胞增殖能力、克隆形成能力和凋亡率。结果肝癌细胞BEL-7402、HepG2、SMMC-7721、MHCC-97H和Hep3B中的miR-143-5p表达明显低于正常肝细胞HL-7702(P<0.01)。与mimics-NC组相比,miR-143-5p mimics组BEL-7402和HepG2细胞增殖能力和克隆形成能力明显降低(P<0.01),凋亡率明显上升(P<0.01)。与mimics-NC组相比,miR-143-5p mimics组BEL-7402和HepG2中Bax和Caspase-3表达明显上升(P<0.01),Bcl-2表达明显下降(P<0.01)。靶基因预测网站和双荧光素酶报告基因验证AGR2是miR-143-5p的靶基因。与mimics-NC组相比,miR-143-5p mimics组靶基因AGR2的表达明显下降(P<0.01)。与si-NC组相比,si-AGR2组BEL-7402和HepG2细胞增殖能力和克隆形成能力明显下降(P<0.01),凋亡率明显上升(P<0.01)。结论miR-143-5p可能通过靶向AGR2抑制肝癌细胞的增殖,促进肝癌细胞的凋亡。Objective To investigate the role of miR-143-5p in the proliferation and apoptosis of hepatocellular carcinoma cells and its mechanism.Methods Real-time quantitative PCR(qRT-PCR)was used to detect the expression of miR-143-5p in normal hepatocytes(HL-7702)and hepatocellular carcinoma cells BEL-7402,HepG2,SMMC-7721,MHCC-97H and Hep3B.Hepatocellular carcinoma cells BEL-7402 and HepG2 were respectively divided into two groups:mimics-NC group(transfected with nonsense sequence mimics-NC)and miR-143-5p mimics group(transfected with miR-143-5p mimics).MTT assay,clone formation assay,flow cytometry and Western blot were used to detect cell proliferation ability,clone formation ability,apoptosis rate and expression of apoptosis-related genes(Bax,Caspase-3 and Bcl-2)in mimics-NC group and miR-143-5p mimics group,respectively.The taeget gene prediction website RNA22 was used to predict the target of miR-143-5p.Luciferase reporter assay was used to identify the targeting relationship between miR-143-5p and anterior gradient 2(AGR2).In BEL-7402 and HepG2 cells,the expression levels of AGR2 in mimics-NC group and miR-143-5p mimics group were detected by qRT-PCR and Western blot.Then,hepatocellular carcinoma cells BEL-7402 and HepG2 were respectively divided into two groups:si-NC group(transfected with nonsense sequence si-NC)and si-AGR2 group(transfected with si-AGR2 mimics).MTT assay,clone formation assay and flow cytometry were used to detect cell proliferation ability,clone formation ability,apoptosis rate in si-NC group and si-AGR2 group.Results The expression levels of miR-143-5p in hepatocellular carcinoma cells BEL-7402,HepG2,SMMC-7721,MHCC-97H and Hep3B were significantly lower than that in normal hepatocytes HL-7702(P<0.01).Compared with mimics-NC group,the cell proliferation ability and the clone formation ability were significantly decreased in miR-143-5p mimics group(P<0.01),while the apoptosis rate was significantly increased(P<0.05).Compared with mimics-NC group,the expression levels of Bax and Caspase-3 were si

关 键 词：肝癌细胞 miR-143-5p 细胞增殖 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]