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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 HBx protein is widely involved in the occurrence and progression of human primary hepatocellular carcinoma (HCC) by regulating the expression of protein coding genes. In this study, the regulatory function of HBx was extended to the non-coding RNA field to explore whether HBx could regulate the expression of microRNA (miRNA) in host hepatocellular carcinoma cells and then participate in the growth and malignant transformation of hepatoma cells. Methods High-throughput microarray analysis and quantitative real-time polymerase chain reaction (q RT-PCR) were used to identify the expression of miRNAs induced by HBx in hepatoma cells. A series of protein and mRNA expression analysis, cell cycle and apoptosis assays and luciferase reporter assays were used to detect the biological changes of HepG2 hepatoma cells after miR-16 family was inhibited. Results The results of microarray analysis showed that HBx induced a large number of miRNAs expression in HepG2 cells. The low expression of miR-16 family was found in HepG2, SK-HEP-1 and Huh7 cells. Meanwhile, HBx significantly up-regulated the expression of CCND1, a target gene of miR-16 family. c-myc mediates silencing of HBx-associated miR-16 family, and exogenous miR-16 / 15a can inhibit the proliferation and clonality of HepG2-hbx (stably expressing HBx) cells by arresting cell cycle progression and inducing apoptosis And non-adherent growth ability. In contrast, silencing miR-16 expression can promote the cycle progression and growth of HepG2 cells. Conclusion HBx can change the miRNA expression profile of hepatocellular carcinoma cells in vitro, especially the silenced miR-16 family. C-myc overexpression mediates HBx down-regulation of miR-15a / 16 and is necessary for HBx to promote the malignant transformation of HepG2 cells. Therefore, miR-16 family may become the target of HBV-related hepatocellular carcinoma.