微小RNA-145-5p对结肠癌细胞增殖凋亡和FSCN1表达的影响  

Effect of microRNA-145-5p on proliferation, apoptosis and expression of FSCN1 in colon cancer cells

作　　者：朱琳[1] 曾庆松[1] 王续[1] ZHU Lin;ZENG Qingsong;WANG Xu(Department of Gastroenterology,the First Affiliated Hospital of Nanyang Medical College,Nanyang 473058,China)

机构地区：[1]南阳医学高等专科学校第一附属医院消化内科

出　　处：《临床肿瘤学杂志》2019年第11期999-1003,共5页Chinese Clinical Oncology

摘　　要：目的探讨微小RNA-145-5p(miR-145-5p)对结肠癌细胞增殖、凋亡和Fascin-1(FSCN1)表达的影响。方法采用实时定量PCR(QPCR)检测人正常结肠上皮NCM460细胞和结肠癌HCT116细胞的miR-145-5p水平;体外常规培养HCT116细胞并分为对照组、过表达组和抑制组,过表达组转染miR-145-5p模拟物mimics,抑制组则转染miR-145-5p抑制物inhibitor,而对照组不作特殊处理。采用QPCR、活细胞计数CCK-8、AnnexinⅤ-FITC/PI双染流式细胞术和Western blotting检测miR-145-5p水平、增殖活力、凋亡率和FSCN1水平;构建FSCN1基因3’端非翻译区(3’UTR)野生型和突变型重组psiCHECK2荧光素酶报告载体并通过双荧光素酶报告基因系统验证miR-145-5p与FSCN1的靶向关系。结果HCT116细胞的miR-145-5p水平为0.281±0.072,低于NCM460细胞的1.062±0.226(P<0.05)。与对照组的1.103±0.214相比,过表达组的miR-145-5p水平升高至4.159±1.080,而抑制组则降低至0.348±0.052;与对照组相比,过表达组转染48、72 h后的增殖活力降低,而抑制组则升高;与对照组的(7.539±1.413)%相比,过表达组的凋亡率升高至(17.126±4.274)%,而抑制组则降低至(2.658±0.725)%;与对照组的0.521±0.078相比,过表达组的FSCN1水平降低至0.243±0.034,而抑制组则升高至0.769±0.092;以上组间差异均有统计学意义(P<0.05)。与转染突变型FSCN13’UTR的荧光素酶报告载体相比,miR-145-5p mimics能降低FSCN13’UTR野生型的荧光素酶活性(P<0.05),但对突变型无影响(P>0.05)。结论miR-145-5p在结肠癌细胞中低表达并发挥抑癌作用,该miRNA可能通过靶向FSCN1来调控癌细胞的增殖并促进其凋亡,有望成为结肠癌的新型生物诊治靶点。Objective To investigate the effect of microRNA-145-5 p(miR-145-5 p)on proliferation,apoptosis and expression of fascin-1(FSCN1)in colon cancer cells.Methods The miR-145-5 p levels of normal human colon epithelium NCM460 cells and colon cancer HCT116 cells were detected by real-time quantitative PCR(QPCR).HCT116 cells were cultured in vitro and divided into control group,overexpression group and inhibition group.The overexpression group was transfected with miR-145-5 p mimics,and inhibition group was transfected with miR-145-5 p inhibitor,while the control group was not treated specially.The miR-145-5 p level,proliferation activity,apoptotic rate and FSCN1 level were detected by QPCR,cell count kit(CCK)-8,AnnexinⅤ-FITC/PI double staining flow cytometry and Western blotting.The recombinant psiCHECK2 luciferase report vector carrying wild and mutant 3’untranslated region(3’UTR)of FSCN1 gene was constructed and the target relationship between miR-145-5 p and FSCN1 was verified by double luciferase reporter gene system.Results The miR-145-5 p level in HCT116 cells was 0.281±0.072,lower than 1.062±0.226 in NCM460 cells(P<0.05).Compared with 1.103±0.214 in control group,the miR-145-5 p level in overexpression group increased to 4.159±1.080,while that in inhibition group decreased to 0.348±0.052;compared with control group,the proliferation activity in the overexpression group decreased at 48 and 72 h after transfection,while those in inhibition group increased;compared with(7.539±1.413)%in control group,the apoptotic rate in overexpression group increased to(17.126±4.274)%,while that in inhibition group decreased to(2.658±0.725)%;compared with 0.521±0.078 in control group,the FSCN1 level in overexpression group decreased to 0.243±0.034,while that in inhibition group increased to 0.769±0.092;the difference among groups was statistically significant(P<0.05).Compared with the luciferase reporter vector carrying the mutant FSCN13’UTR,miR-145-5 p mimics could reduce the luciferase activity of the wild typ

关 键 词：结肠癌 微小RNA-145-5p 增殖凋亡 FASCIN-1 

分 类 号：R735.3[医药卫生—肿瘤]