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【目的】在细胞水平上研究乙型肝炎病毒X蛋白(HBx)对胰岛素样生长因子结合蛋白3(IGFBP3)转录的影响并对具体机制进行初步探索。【方法】首先采用RNA-Deep-Sequencing技术分析HepG2和乙型肝炎病毒(HBV)转基因细胞HepG2-4D14中表达差异的基因,然后通过实时定量PCR对相关基因进行验证;利用启动子报告基因分析,研究HBx对相关基因IGFBP3转录的调控;通过染色质免疫共沉淀方法,分析HBx抑制IGFBP3启动子活性的机制。【结果】RNA-Deep-Sequencing的结果表明IGFBP3在HBV转基因细胞HepG2-4D14中水平显著下调,实时定量PCR结果与RNA-Deep-Sequencing一致。进一步研究表明HBx能明显抑制IGFBP3的转录,通过实时定量PCR发现HBx对IGFBP3转录的抑制作用依赖于p53;染色质免疫共沉淀实验结果表明HBx能够通过抑制p53与IGFBP3启动子的结合,从而抑制IGFBP3的转录。IGFBP3是一种细胞周期负调控蛋白,我们推测HBx对IGFBP3水平的下调是其促进细胞增殖的途径之一。【结论】HBV能显著下调IGFBP3的转录,机制研究揭示HBV HBx通过干扰p53与IGFBP3启动子的结合进而抑制IGFBP3的转录。
【Objective】 To investigate the effects of hepatitis B virus X protein (HBx) on the transcription of insulin-like growth factor binding protein-3 (IGFBP3) at the cellular level and to explore the specific mechanism. 【Methods】 The genes of HepG2 and HepG2-4D14 in Hepatitis B virus (HBV) transgenic cell line HepG2 were analyzed by RNA-Deep-Sequencing. The related genes were verified by real-time quantitative PCR. Using the promoter reporter gene analysis, To study the regulation of HBx on IGFBP3 transcription of related genes and to investigate the mechanism of HBx inhibiting IGFBP3 promoter activity by chromatin immunoprecipitation. [Results] The results of RNA-Deep-Sequencing showed that the level of IGFBP3 was significantly down-regulated in HepG2-4D14 HBV transgenic cells. The results of real-time quantitative PCR were consistent with that of RNA-Deep-Sequencing. Further studies showed that HBx can significantly inhibit the transcription of IGFBP3, and HBx can inhibit the transcription of IGFBP3 by real-time quantitative PCR. The results of chromatin immunoprecipitation showed that HBx could inhibit the binding of IGFBP3 to IGFBP3 by inhibiting the binding of p53 to IGFBP3 Of transcription. IGFBP3 is a negative regulator of the cell cycle. We hypothesize that the down-regulation of IGFBP3 by HBx is one of the ways to promote cell proliferation. 【Conclusion】 HBV can significantly down-regulate the transcription of IGFBP3. Mechanism studies reveal that HBV HBx inhibits the transcription of IGFBP3 by interfering with the binding of p53 to the IGFBP3 promoter.