HBx下调miR-16家族表达促进肝癌细胞恶性转化  

HBx promotes hepatoma cell malignant transformation via repressing miR-16 family

作　　者：伍刚[1] 郑波[1] 黄锐[1] 杨训[1] 

机构地区：[1]四川省人民医院肝胆外科,成都610072

出　　处：《中国普外基础与临床杂志》2017年第4期412-419,共8页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金(项目编号:81302161);四川省卫计委科研课题(项目编号:150215)

摘　　要：目的乙型肝炎病毒X(hepatitis B virus X protein,HBx)蛋白通过调控蛋白编码基因表达而广泛参与人原发性肝细胞癌(简称肝癌)的发生和进展。本研究将HBx的调控功能拓展到非编码RNA领域,探讨HBx能否调控宿主肝癌细胞内microRNA(miRNA)的表达,进而参与肝癌细胞生长及恶性转化。方法利用高通量芯片分析及实时定量聚合酶链反应(quantitative real time polymerase chain reaction,q RT-PCR)鉴定HBx在肝癌细胞内诱发的miRNA表达变化。通过系列蛋白和mRNA表达分析,细胞周期及凋亡实验以及萤光素酶报告实验来检测miR-16家族表达抑制后HepG2肝癌细胞的生物学变化。结果芯片分析结果显示,HBx在HepG2细胞内诱发了大量的miRNAs表达变化,其中miR-16家族的低表达能在HepG2、SK-HEP-1及Huh7肝癌细胞中得到重复。同时,HBx显著上调miR-16家族的靶基因CCND1的表达。c-myc介导了HBx相关miR-16家族沉默,外源表达的miR-16/15a能通过阻滞细胞周期进展和诱导凋亡而抑制HepG2-hbx(稳定表达HBx)细胞的增殖、克隆形成及非贴壁生长能力。反之,沉默miR-16表达能促进HepG2细胞的周期进展和生长。结论 HBx能在体外改变肝癌细胞的miRNA表达谱,尤其是沉默miR-16家族表达。c-myc高表达介导了HBx下调miR-15a/16的过程且是HBx促进HepG2细胞恶性转化所必须的。因此,miR-16家族可能成为HBV相关肝细胞癌的治疗靶点。Objective Hepatitis B virus X （HBx） protein is involved in the initiation and progression of hepatocellular carcinoma （HCC） by regulating the host protein-coding genes. Herein, we want to explore whether HBx protein can alter the expression of microRNAs （miRNAs） to promote proliferation and transformation in malignant hepatocytes in vitro. Methods MiRNA microarray and quantitative reverse-transcription polymerase chain reactions （qRT-PCRs） were performed to identify miRNAs that were differentially regulated by HBx protein in HCC cells. Protein and mRNA expression analyses, cell cycle and apoptosis analyses, and luciferase reporter assays were performed to delineate the consequences of miR-16 family repression in HepG2 cells. Results HBx protein induced widespread deregulation of miRNAs in HepG2 cells, and the downregulation of the miR-16 family was reproducible in HepG2, SK- HEP-1, and Huh7 cells. CCND 1, a target gene of the miR-16 family, was derepressed by HBx protein in HepG2 cells. C-myc mediated the HBx-induced repression of miR-15a/16 in HepG2 cells. Ectopically expressed miR-15a/16 suppressed the proliferation, clonogenicity, and anchorage-independent growth of HBx-expressing HepG2 ceils by arresting them in the G1 phase and inducing apoptosis, whereas reduced expression of miR-16 accelerated the growth and cell-cycle progression of HepG2 ceils. Conclusions HBx protein altered the in vitro expression of miRNAs in host malignant hepatocytes, particularly downregulating the miR-16 family. Repression of miR-15a/16 is c-myc mediated and is required for the HBx-induced transformation of HepG2 cells in vitro. Therefore, miR-16 family may serve as a therapeutic target for hepatitis B virus （HBV）-associated HCC.

关 键 词：乙型肝炎病毒X蛋白 miRNA miR-16家族 C-MYC 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]