miR-519d通过靶向基因表达抑制非小细胞肺癌增殖作用及其机制的研究  

miR-519d suppresses proliferation of non-small cell lung cancer cells by targeting CCR-7

作　　者：孙静 杨明 刘祯璐 王妍 蒋雨涵 谢良军[1,3] 孙辉[1,4] Sun Jing;Yang Ming;Liu Zhenlu;Wang Yan;Jiang Yuhan;Xie Liangjun;Sun Hui(College of Pharmacy,Harbin Medical University,Harbin 150081,China;School of Basic Medicine,Harbin Medical University,Harbin 150081,China;Academic Administration,College of Pharmacy,Harbin Medical University,Harbin 150081,China;Pharmaceutical Experiment Teaching Center,College of Pharmacy,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学药学院,150081 [2]哈尔滨医科大学基础医学院,150081 [3]哈尔滨医科大学药学院教务科,150081 [4]哈尔滨医科大学药学实验教学中心,150081

出　　处：《国际遗传学杂志》2020年第2期82-89,共8页International Journal of Genetics

基　　金：黑龙江省大学生创新创业训练计划项目(201810226069)。

摘　　要：目的探究miR-519d对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖作用的影响及其潜在的作用机制。方法通过对芯片GSE19945的中NSCLC癌和癌旁组织中不同miRNA的差异表达,Starbase(http://starbase.sysu.edu.cn)数据分析CC趋化因子受体7(CC chemokine receptor 7,CCR-7)在NSCLC患者中的表达,预后miR-519d以及CCR-7两者的相关性。生物信息方法miR-519d与CCR-7潜在的结合位点。实时定量PCR检测A549细胞中转染miR-519d后miR-519d的表达;通过CCK-8检测不同表达miR-519d对于A549细胞活性的影响,克隆形成以及Ki67检测过表达或者低表达miR-519d后对于A549细胞增殖能力的影响。荧光素酶报告验证CCR-7与miR-519d的靶向作用。采用应用免疫蛋白印迹(Western blot)检测不同表达miR-519d后A549细胞中PCNA、Bax以及CCR-7蛋白的表达。结果对于芯片GSE19945结果分析,发现miR-519d在癌症组织中明显低表达(P=0.045),在NSCLC中miR-519d表达明显高于正常组织;低表达miR-519d的患者生存率明显高于高表达miR-519d的NSCLC患者(P=0.009),同时,CCR-7在NSCLC患者组织中的表达明显升高,低表达CCR-7的患者生存率明显高于过表达CCR-7的NSCLC患者,且miR-519d与CCR-7在NSCLC患者中表达呈负相关。高表达miR-519d后,A549细胞活性相对于阴性对照组明显降低(P=0.043),而低表达miR-519d后活性明显升高(P=0.031)。过表达miR-519d后A549细胞增殖能力相对于阴性对照组明显降低。生物信息学结果显示miR-519d与CCR-7有潜在的结合位点。Western blot结果表明,不同表达的miR-519d能够影响增殖相关蛋白PCNA以及Bax表达(P=0.029、0.031)。过表达miRNA-519d后CCR-7表达相对于阴性对照组明显降低(P=0.038、0.027),而低表达miRNA-519d后,CCR-7表达明显升高(P=0.043、0.030),同时荧光素酶报告结果显示,CCR-7是miRNA-519d的直接作用靶点(P=0.031)。结论miR-519d能通过靶向调控CCR-7基因的表达进而调控NSCLC细胞增殖,同时miR-519d也是Objective To investigate the effect of miR-519d and CC chemokine receptor 7(CCR-7)on the proliferation of non-small cell lung cancer(NSCLC)cells and their potential underlying mechanisms.Methods The expression,prognosis and correlation of miR-519d and CCR-7 in patients with NSCLC were analyzed by Starbase(http://starbase.sysu.edu.cn)data.Potential binding sites between miR-519d and CCR-7 were identified by bioinformatics method.The expression of miR-519d in A549 cells transfected with miR-519d was detected by real-time quantitative PCR.CCK-8 was used to detect the effect of different expression of miR-519d on the activity of A549 cells,clone formation and Ki67 to detect the effect of overexpression or low expression of miR-519d on the proliferation of A549 cells.Luciferase report confirmed the targeting effect of CCR-7 and miR-519d.Immunoblotting(Western blot)was used to detect the expression of PCNA,Bax and CCR-7 protein in A549 cells after different expression of miR-519d.Results For the analysis of GSE19945,miR-519d was significantly down-regulated in cancer tissues compared to normal group(P=0.045).The survival rate of patients with low expression of miR-519d was significantly higher than that of patients with high expression of miR-519d(P=0.009).At the same time,the expression of CCR-7 was significantly increased in patients with NSCLC.The survival rate of patients with low expression of CCR-7 was significantly higher than that of NSCLC patients with overexpression of CCR-7.There was a negative correlation between the expression of miR-519d and CCR-7 in NSCLC patients.After high expression of miR-519d,the activity of A549 cells decreased significantly compared with the negative control group,while the activity of A549 cells increased significantly after low expression of miR-519d.The proliferation ability of A549 cells after overexpression of miR-519d was significantly lower than that of the negative control group(P=0.031).Bioinformatics results showed that miR-519d has a potential binding site with CCR-7.We

关 键 词：miR-519d CC趋化因子受体7 非小细胞肺癌 增殖 

分 类 号：R734[医药卫生—肿瘤]