miR-383靶向Notch1对结肠癌HCT116细胞增殖和迁移的影响  

Effects of miR-383 targeting Notch1 on proliferation and migration of colon cancer HCT116 cells

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 Tumor 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：《国际消化病杂志》2021年第1期38-44,共7页International Journal of Digestive Diseases

基　　金：2019年度河北省医学课题研究计划(20190914)。

摘　　要：目的分析miR-383和Notch1在结肠癌中的作用及机制。方法选择2017年4月至2020年3月在河北北方学院附属第一医院经病理检查确诊为结肠癌的74例患者,收集其经手术切除的肿瘤组织及癌旁正常组织(距肿瘤边缘>3 cm)。采用TargetScan预测miR-383的潜在靶基因,采用双荧光素酶报告基因实验、逆转录定量聚合酶链反应(qRT-PCR)和蛋白质印迹法(Western Blot)验证这一预测。采用免疫组织化学法、Western Blot和qRT-PCR检测Notch1在结肠癌组织和癌旁正常组织中的表达水平。分别在结肠癌HCT116细胞中转染阴性对照(NC)、miR-383模拟物、Notch1及共转染miR-383模拟物+Notch1,对应分为miR-NC组、miR-383组、Notch1组及miR-383+Notch1组;另设一组空白对照组。采用CCK8法和Tranwell实验检测miR-383模拟物和Notch1对结肠癌细胞增殖和迁移能力的影响。构建结肠癌裸鼠移植瘤模型,将裸鼠随机分为miR-NC模型组(n=3)和miR-383模型组(n=3),观察miR-383模拟物对裸鼠肿瘤生长的影响。结果双荧光素酶报告基因实验结果显示,miR-383组的Notch1野生型质粒荧光素酶活性较miR-NC组明显降低(20.07±0.12比13.61±0.19,P<0.01)。与miR-NC组相比,miR-383组中Notch1 mRNA和蛋白表达均明显降低(P均<0.01)。结肠癌组织中的Notch1 mRNA和蛋白表达水平均明显高于癌旁正常组织(P均<0.01)。细胞实验显示,与miR-NC组相比,Notch1组细胞的增殖、迁移能力明显升高,miR-383组细胞的增殖、迁移能力降低;miR-383+Notch1组的细胞增殖、迁移能力较miR-383组明显升高。在结肠癌移植瘤裸鼠模型中,miR-383模型组肿瘤组织中Ki67和Notch1蛋白表达水平均明显低于miR-NC模型组。结论Notch1具有促进结肠癌细胞增殖和迁移的作用,而miR-383能靶向调控Notch1以抑制结肠癌细胞的生物学行为并减缓裸鼠肿瘤的生长。miR-383具有抑制结肠癌的作用。Objective This paper intends to analyze the role and mechanism of miR-383 and Notch1 in colon cancer.Methods From April 2017 to March 2020 in the First Affiliated Hospital of Hebei North University,74 patients who were diagnosed with colon cancer by pathological examination were selected,and their surgically resected tumor tissues and normal adjacent tissues(more than 3 cm from the adge of the tumor)were collected.TargetScan was used to predict the potential target genes of miR-383,and the prediction was verified by double luciferase reporter gene assay,quantitative reverse transcription polymerase chain reaction(qRT-PCR),and Western Blot.Immunohistochemistry,Western Blot and qRT-PCR were used to detect the expressions of Notch1 in colon cancer tissues and normal adjacent tissues.The negative control(NC),miR-383 mimic,Notch1,and co-transfect miR-383 mimic+Notch1 were transfect into colon cancer HCT116 cells,respectively,which were divided into the miR-NC group,the miR-383 group,the Notch1 group,and the miR-383+Notch1 group.In addition,the control group was established.CCK8 and tranwell assay were used to detect the effects of miR-383 mimic and Notch1 on the proliferation and migration of colon cancer cells.A nude mouse xenograft model of colon cancer was constructed,and nude mice were randomly assigned to the miR-NC model group(n=3)and the miR-383 model group(n=3),and the effects of miR-383 mimics on tumor growth in nude mice were observed.Results The results of the double luciferase reporter gene assay showed that the Notch1 wild-type plasmid luciferase activity of the miR-383 group was significantly lower than that of the miR-NC group(20.07±0.12 vs.13.61±0.19,P<0.01).Compared with the miR-NC group,the expression of Notch1 mRNA and protein in the miR-383 group was significantly reduced(both P<0.01).The expressions of Notch1 mRNA and protein in colon cancer tissues were significantly higher than those in normal tissues adjacent to cancer(both P<0.01).Cell experiments showed that compared with the miR-NC group,t

关 键 词：结肠癌 miR-383 NOTCH1 

分 类 号：R735.35[医药卫生—肿瘤]