知识与财富的链接
【目的】分析丙型肝炎病毒(HCV)核心蛋白(CORE)稳定表达对磷酸烯醇式丙酮酸羧基酶(PCK1)转录水平的影响,并分析HCV CORE调控PCK1转录的分子机制,为进一步阐明HCV感染致2型糖尿病机理的探讨提供新的思路。【方法】利用反转录病毒表达系统构建稳定表达HCV CORE的Huh7-lunet-core细胞系。采用Real-time PCR和萤光素酶报告基因技术检测Huh7-lunet-core细胞系中PCK1、FOXO1以及PGC-1α转录水平变化,并结合Western blot分析FOXO1的活性变化。【结果】HCV CORE的稳定表达显著增强PCK1的转录水平,HCV CORE不影响FOXO1的转录和表达水平,但降低FOXO1的磷酸化水平,激活了FOXO1的转录活性,并增强PGC-1α的mRNA表达水平。【结论】HCV CORE在Huh7-lunet细胞中的稳定表达激活FOXO1的转录活性,并与PGC-1α协同作用,上调PCK1的转录,从而导致肝糖异生过度发生,对HCV CORE调控PCK1转录的分子机制的揭示可能为HCV感染相关的糖尿病的治疗提供新的靶点。
[Objective] We analyzed the effect of the stable expressed Hepatitis C virus core protein on PCK1 mRNA expression level and the molecular mechanisms involved in Huh7-lunet cells.[Methods] A retroviral vector mediated mammalian cell expression cell line of the HCV core protein was constructed.The mRNA and protein levels of PCK1,FOXO1 and PGC-1α were analyzed by Real-time PCR and luciferase assay in Huh7-lunet-core cells.[Results] HCV CORE upregulated the mRNA levels of PCK1 significantly.Both the mRNA and protein levels of FOXO1 were not affected in Huh7-lunet-core cells,whereas a decreased phosphorylation status of FOXO1 was exhibited.Moreover,activation of FOXO1 by HCV CORE was detected.Further,the mRNA level of PGC-1α was found to be significantly elevated in Huh7-lunet-core cells.[Conclusion] Our results revealed for the first time that HCV core protein expression-mediated FOXO1 activation and the increased PGC-1α leaded to the elevation of PCK1 at the mRNA level,which suggesting the immoderate gluconeogenesis in HCV-infected hepatocytes.Our findings contributed to the understanding of the molecular mechanisms of HCV-related insulin resistance and provided potential new clues for the prevention and therapy of diabetes.
