HCV核心蛋白调控SREBP-1c表达水平的机制研究

Molecular mechanism of SREBP-1c expression regulated by HCV core protein

目的探讨HCV核心蛋白调控SREBP-1c的分子生物学机制。方法构建HCV核心蛋白真核表达载体pc DNA3.1/myc-His(-)-Core(1b,3a),转染Hep G2细胞系,48 h后提取细胞总RNA和总蛋白,采用real-time PCR、Western blot等方法检测脂代谢相关基因Sirt1和SREBP-1c的mRNA和蛋白表达水平;构建Sirt1启动子报告基因表达载体p GL4.10-Sirt1,与pc DNA3.1/myc-His(-)-Core(1b,3a)共转染Hep G2细胞系24 h后检测核心蛋白对Sirt1启动子活性的影响;构建pmir GLO-SREBP-1c-3'-UTR报告基因表达载体,与pc DNA3.1/myc-His(-)-Core(3a)共转染Hep G2细胞系48 h后检测SREBP-1c-3'-UTR相对luciferase活性。结果与对照组相比,1b型和3a型HCV核心蛋白表达组SREBP-1c的mRNA表达上调(分别上调1.358倍和1.337倍,P=0.043、0.008)SREBP-1c蛋白表达水平上调(分别上调1.608倍和1.926倍,P=0.042、0.008);与对照组相比,1b型和3a型HCV核心蛋白表达组Sirt1 mRNA表达上调(分别上调1.566倍和1.71倍,P=0.037、0.006),Sirt1蛋白表达水平上调(分别上调1.436倍和1.588倍,P=0.026、0.009);与对照组相比,HCV核心蛋白表达组Sirt1的启动子活性无显著变化;与对照组相比,HCV核心蛋白表达组SREBP-1c-3'-UTR相对luciferase活性下调(相对值为0.667,P=0.008)。结论 HCV核心蛋白上调SREBP-1c与Sirt1的表达,可能是HCV相关性肝脂肪变的发病机制之一。micro RNA参与HCV核心蛋白对SREBP-1c的表达调控,而是否有micro RNA对HCV调控Sirt1表达发挥作用尚待于进一步的研究证实。

Objective To investigate the molecular mechanism of SREBP-1c expression regulated by HCV core protein. Methods HepG2 cells were transfected with pcDNA3.1/myc-His（-）-Core（1b, 3a）, and the mRNA and protein expression levels of SREBP-1c and Sirt1 were determined by real time PCR and Western blot 48 h post-transfection. pGL4.10-Sirt1 and pcDNA3.1/myc-His（-）-Core（1b, 3a） were co-transfected into HepG2 cells, the Sirt1 promoter activity was measured by Luciferase assay 24h post-transfection. pmirGLO-SREBP-1c-3＇-UTR and pcDNA3.1/myc-His（-）-Core （3a） were co-transfected into HepG2 cells, and the relative luciferase activity of SREBP-1c-3＆#39;-UTR was measured by Luciferase assay 48h post-transfection. Results HCV core proteins （1b, 3a） up-regulate the mRNA levels （1.358 times and 1.337 times respectively;P = 0.043, 0.008） and protein expression （1.608 times and 1.926 times respectively, P = 0.042, 0.008） of SREBP-1c. Both HCV core proteins of genotype 1b and 3a up-regulate the mRNA levels （1.566 times and 1.71 times respectively; P = 0.037, 0.006） and protein expression （1.436 times and 1.588 times respectively, P =0.026, 0.009） of Sirt1. The Sirt1 promoter did not show significant transcriptional activity in the presence of HCV core proteins （1b, 3a）. The relative luciferase activity of SREBP-1c-3＆#39;-UTR shown significant decrease in the presence of HCV core protein of genotype 3a （RQ = 0.667, P = 0.008）. Conclusions HCV core＆amp;nbsp;proteins up-regulate the expression levels of SREBP-1c and Sirt1, which may be one of the pathogenesis of HCV related steatosis. MicroRNA also involved in the regulation of SEBP-1c by HCV core protein. Whether there are microRNAs contribute to the regulation of Sirt1 by HCV core protein still need to be investigated.