HBx抑制IGFBP3转录的机制

Mechanism of the inhibitory effect of HBx on IGFBP3 transcription

摘要:

[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。

Abstract:

[Objective] To study the mechanism of HBx regulating the transcription of insulin growth factor binding protein 3 (IGFBP3).[Methods] RNA-sequencing method was used to screen differently expressing genes in HepG2 and HBV transgenic cell line HepG2-4D14. The mRNA of IGFBP3 was measured by reverse transcription and real-time PCR. To verify the activity of IGFBP3 promoter, cells were analyzed by luciferase assay. The binding of p53 and IGFBP3 promoter was measured by ChIP Assay.[Results] The level of IGFBP3 mRNA in HBV transgenic cell line HepG2-4D14 was significantly lower than that in HepG2 cells. The data of real-time PCR indicated that HBV HBx can down regulate the transcription of IGFBP3. By taking the approach of promoter luciferase assay on HCT116 and HCT116-p53^-/- cell lines, we found that HBx can inhibit the promoter activity of IGFBP3 in a p53-dependent manner. Our data also showed that HBx can significantly interfere with the binding of p53 to the promoter of IGFBP3. As IGFBP3 is a suppressor for cell growth, we postulate that HBx promotes the cell proliferation by reducing the level of IGFBP3.[Conclusion] HBx can inhibit the transcription of IGFBP3 in a p53-dependent manner.