微小RNA-375调控自噬抑制肝癌细胞SMMC-7721增殖和转移  

MicroRNA-375 inhibited proliferation and metastasis of hepatoma cell line SMMC-7721 by regulating autophagy

作　　者：王星星 宋虎[1] 杜晨阳[1] 王振[2] 张建军[2] 沈中阳[2] WANG Xing-xing;SONG Hu;DU Chen-yang;WANG Zhen;ZHANG Jian-jun;SHEN Zhong-yang(The First Central Clinical College of Tianjin Medical University,Tianjin 300192,China;Liver Transplantation Department,Tianjin First Central Hospital)

机构地区：[1]天津医科大学一中心临床学院,300192 [2]天津市第一中心医院移植外科

出　　处：《天津医药》2018年第12期1267-1272,1370,共7页Tianjin Medical Journal

基　　金：天津市器官移植临床医学研究中心项目(15ZXLCSY00070)

摘　　要：目的探讨微小RNA-375(miR-375)通过调控自噬相关蛋白(Atg)14介导的自噬对肝癌细胞(SMMC-7721)增殖和转移能力的影响。方法将SMMC-7721细胞分别用miR-375模拟物、抑制物以及Atg14的干扰RNA处理,并建立缺氧1 h复氧6 h的模型,分为miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组以及siRNA NC组、Atg14 siRNA组、miR-375+Atg14 siRNA组。TargetScan预测miR-375与Atg14基因相关性。荧光实时定量PCR(qRT-PCR)检测miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组miR-375、Atg14 m RNA相对表达量。免疫细胞化学染色检测3组细胞内N-钙黏素(N-Cadherin)和β-连环蛋白(β-catenin)表达分布情况。绿色荧光蛋白-红色荧光蛋白-轻链(mGFP-RFP-LC3)自噬双标腺病毒转染3组细胞,荧光显微镜下观察自噬体形成情况。平板克隆检测肝癌细胞增殖情况。Western blot检测Atg14、泛素结合蛋白(P62)、轻链3Ⅰ(LC3Ⅰ)、轻链3Ⅱ(LC3Ⅱ)、酵母Atg6同源物(Beclin1)、N-Cadherin、β-catenin和波形蛋白(Vimentin)表达情况。结果 miR-375与Atg14基因相关性较高,qRT-PCR显示过表达miR-375能抑制Atg14 mRNA表达;反之能促进Atg14 mRNA表达。过表达miR-375能抑制肝癌细胞内N-Cadherin表达、提高β-catenin表达水平,抑制肝癌细胞增殖能力(P<0.01);而抑制miR-375表达能促进N-Cadherin表达、降低β-catenin表达水平,提高肝癌细胞增殖能力(P<0.01)。过表达miR-375能抑制Atg14、LC3Ⅱ、Beclin1表达,促进P62表达(P<0.01);反之能促进Atg14、LC3Ⅱ、Beclin1表达,抑制P62表达(P<0.01)。荧光显微镜下观察到过表达miR-375可抑制自噬体形成,而抑制miR-375表达能促进自噬体形成(P<0.01)。干扰Atg14表达后可增强miR-375对肝癌细胞增殖、转移能力的抑制作用(P<0.01)。结论激活miR-375能抑制肝癌细胞增殖和转移,而抑制miR-375能促进肝癌细胞增殖和转移,其机制可能与miR-375通过靶向调控Atg14抑制自噬,从而抑制SMMC-7721肝癌细胞增殖和转移有�Objective To investigate the effect of microRNA-375(miR-375)targeting autophagy associated gene14(Atg14)-mediated autophagy on the proliferation and metastasis of hepatoma cells(SMMC-7721).Methods SMMC-7721cells were divided into miR-375mimics,miR-375inhibitor and Atg14interfering RNA treatment groups.Models with hypoxia for1h and reoxygenation for6h were established,and were divided into miR-375NC group,miR-375mimics group,miR-375inhibitor group,siRNA NC group,Atg14siRNA group and miR-375+Atg14siRNA group.Targetscan was used to predict the genetic association between miR-375and the Atg14.Fluorescence real-time quantitative PCR(QRTPCR)was used to detect the relative expressions of miR-375and ATG14mRNA in miR-375NC group,miR-375mimics group and miR-375inhibitor group.Immunocytochemical staining was used to detect the expressions of N-Cadherin andβ-catenin in the above three groups.The three groups of cells were transfected with GFP-RFP-LC3adenovirus,and the formation of autophagosomes was observed by the fluorescence microscope.Plate cloning was used to detect the proliferationof liver cancer cells.The expressions of Atg14,P62,LC3Ⅰ,LC3Ⅱ,Beclin1,N-Cadherin,β-catenin and Vimentin weredetected by Western blot assay.Results miR-375gene was highly associated with the Atg14gene.qRT-PCR showed thatoverexpression of miR-375inhibited Atg14mRNA expression,and conversely,it promoted Atg14mRNA expression.Overexpression of miR-375could inhibit the expression of N-Cadherin,increase the expression ofβ-catenin,and inhibit theproliferation of hepatoma cells(P<0.01).On the contrary,the inhibition of miR-375expression promoted the expression ofN-Cadherin,decreasedβ-catenin expression and increased the proliferation of liver cancer cells(P<0.01).Overexpressionof miR-375inhibited the expression levels of Atg14,LC3Ⅱand Beclin1,and promoted the expression of P62(P<0.01).Conversely,it promoted the expressions of Atg14,LC3Ⅱand Beclin1,and inhibited the expression of P62(P<0.01).Byfluorescence microscope,the inhibition of autop

关 键 词：癌 肝细胞 自噬 微RNAS 细胞增殖 肿瘤转移 

分 类 号：R735.7[医药卫生—肿瘤]