miRNA-541-5p负调控细胞周期蛋白D1抑制结肠癌细胞增殖和迁移的相关研究  

miRNA-541-5p inhibits proliferation and migration of colon cancer cells by negatively regulating cyclin D1

作　　者：王晓元[1] 赵轶峰[1] 杨永江[1] 黄迪[1] 苏卓彬[1] 李坤[2] 李晶晶 李曙光[1] Wang Xiaoyuan;Zhao Yifeng;Yang Yongjiang;Huang Di;Su Zhuobin;Li Kun;Li Jingjing;Li Shuguang(Department of Gastrointestinal Surgery,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China;Department of Pathology,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院附属第一医院胃肠肿瘤外科,河北张家口075000 [2]河北北方学院附属第一医院病理科,河北张家口075000

出　　处：《肿瘤研究与临床》2021年第5期321-327,共7页Cancer Research and Clinic

基　　金：河北省医学课题研究计划(20190914)。

摘　　要：目的探讨miRNA-541-5p(miR-541-5p)负调控细胞周期蛋白D1(CCND1)对结肠癌细胞增殖和迁移的影响及其相关机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-541-5p在结肠癌细胞株HT29、SW480、SW620、HCT116和正常人肠上皮细胞株HIEC以及2017年4月至2020年3月河北北方学院附属第一医院112例结肠癌手术患者肿瘤组织和癌旁正常组织中的表达水平。采用TargetScan生物信息学软件预测miR-541-5p的潜在靶基因,并通过双荧光素酶报告基因实验、qRT-PCR和蛋白质印迹法进行验证。检测CCND1在结肠癌细胞株和结肠癌组织中的表达水平。将miR-541-5p表达水平最低的细胞分为miR-NC组(转染对照质粒)、miR-541-5p组(转染miR-541-5p模拟物)、miR-541-5p+CCND1组(共转染miR-541-5p模拟物和CCND1),使用CCK-8法、Transwell法检测miR-541-5p和CCND1对结肠癌细胞增殖、迁移能力的影响。通过构建结肠癌裸鼠移植瘤模型观察miR-541-5p对肿瘤生长的影响。结果结肠癌患者肿瘤组织中miR-541-5p相对表达量低于癌旁正常组织(0.45±0.06比1.00±0.12,t=43.385,P<0.01)。结肠癌细胞株HT29、SW480、SW620、HCT116和正常人肠上皮细胞株HIEC中的miR-541-5p相对表达量分别为0.46±0.03、0.67±0.04、0.57±0.06、0.17±0.02和1.00±0.15,差异有统计学意义(F=5.621,P<0.01),各结肠癌细胞株miR-541-5p相对表达量均低于正常人肠上皮细胞株HIEC。选择HCT116细胞进行后续实验。TargetScan软件预测CCND1的3'UTR可能存在与miR-541-5p互补位点。双荧光素酶报告基因实验结果显示,CCND1是miR-541-5p的潜在靶基因,miR-541-5p可负调控CCND1表达。CCK-8法检测结果显示,miR-NC组、miR-541-5p组、miR-541-5p+CCND1组HCT116细胞增殖率分别为(2.00±0.16)%、(0.89±0.08)%、(2.56±0.23)%,差异有统计学意义(F=6.715,P<0.01),在miR-541-5p过表达的HCT116细胞中转染CCND1能逆转miR-541-5p对细胞增殖的抑制作用。Transwell检测结果显示,过表达miObjective To investigate the effect of cyclin D1(CCND1)negatively regulated by miRNA-541(miR-541-5p)on the proliferation and migration of colon cancer cells as well as its related mechanism.Methods Expression levels of miR-541-5p in colon cancer cell lines HT29,SW480,SW620,HCT116 and enterocyte line HIEC of the normal people as well as cancer tissues and pericarcinomatous normal tissues of 112 patients undergoing the colon cancer surgery from the First Affiliated Hospital of Hebei North University between April 2017 and March 2020 were detected by using quantitative real-time polymerase chain reaction(qRT-PCR).The potential target gene of miR-541-5p was predicted by using TargetScan,and was verified by using dual luciferase reporter gene assay,qRT-PCR and Western blot.Expression level of CCND1 was detected in colon cancer cell lines and tissues.Cells with the lowest expression level of miR-541-5p were divided into miR-NC group(the transfected control plasmid),miR-541-5p group(the transfected miR-541-5p mimics),miR-541-5p+CCND1 group(the co-transfected miR-541-5p mimics and CCND1).Effect of miR-541-5p and CCND1 on proliferation and migration ability of colon cancer cells was detected by using cell counting kit-8(CCK8)and Transwell method.The xenograft model of colon cancer in nude mice was constructed to observe the effect of miR-541-5p on tumor growth.Results The relative expression level of miR-541-5p in colon cancer tissues was lower than that in pericarcinomatous normal tissues(0.45±0.06 vs.1.00±0.12,t=43.385,P<0.01).The relative expression level of miR-541-5p was 0.46±0.03,0.67±0.04,0.57±0.06,0.17±0.02,1.00±0.15,respectively in colon cancer cell lines HT29,SW480,SW620,HCT116 and enterocyte line HIEC of the normal people,and the difference was statistiacally significant(F=5.621,P<0.01);the relative expression level of miR-541-5p in all colon cancer cell lines was lower than that in enterocyte line HIEC of the normal people.HCT116 cells were selected to make the subsequent experiments.The predicted resul

关 键 词：结肠肿瘤 微RNAS 细胞周期蛋白D1 细胞增殖 细胞迁移分析 

分 类 号：R735.35[医药卫生—肿瘤]