HCV NS3对IFN-λⅠ表达的影响及其调控机制

Effects and regulation mechanisms of HCV NS3 on expression of IFN\|λⅠ in HepG2 cells

目的探讨丙型肝炎病毒(HCV)NS3对干扰素-λⅠ(IFN-λⅠ/IL-29)表达的影响及其调控机制。方法将HCV NS3表达载体pcDNA3.1/myc-His-NS3转染至HepG2细胞,验证HCV NS3蛋白表达之后,采用实时荧光定量PCR、Westernblot和ELISA法观察HCV NS3对IFN-λⅠmRNA及其蛋白表达水平的影响。构建IFN-λⅠ全长启动子报告基因表达载体和3’-非翻译区(3’-UTR)报告基因表达载体,借助双萤光素酶活性检测,探索HCV NS3对IFN-λⅠ转录水平的调控机制。结果 pcDNA3.1/myc-His-NS3在HepG2细胞中成功表达,与转染pcDNA3.1/myc-His空载体相比,pcDNA 3.1/myc-His-NS3过表达时在mRNA和蛋白水平均能抑制HepG2细胞内IFN-λⅠ的表达,差异具有统计学意义(P<0.05)。双萤光素酶活性检测结果显示,与对照组相比,转染IFN-λⅠ全长启动子和3’-非翻译区报告基因表达质粒后,双萤光素酶活性变化无统计学意义(P>0.05)。结论 HCV NS3在mRNA及蛋白水平能抑制IFN-λⅠ的表达,但其转录水平不受影响,具体调控机制尚有待进一步研究。

Objective To investigate on the effects and regulation mechanisms of HCV NS3 on IFN-λI （IL-29） in HepG2 cells. Methods HCV NS3 protein expressive vector was cloned （pcDNA3. l/ myc-His-NS3） and expressed in HepG2 cells, then the expression level of IFN-λI was detected by qRT-PCR, Western blot and ELISA. IFN-λI promoter reporter vector （pGIA. 10-IFN-λI P） as well as the IFN-λI 3＇ -untranslated region （3＇-UTR） reporter vector （pmirGLO-IFN-λ I 3＇-UTR） were constructed and used for studying the regulation mechinasm by luciferase assay. Results HCV NS3 protein expressed in HepG2 cells post-transfection successfully. Both the mRNA and protein expression levels were significantly decreased in the presence of HCV NS3 protein （P 〈 0.05）. However, the results of iuciferase assay showed that HCV NS3 protein had no influence on IFN-λ I promoter activity. In addition, miRNAs were involved in the regulation of IFN-λI by HCV NS3 protein. Conclusions Overexpression of HCV NS3 could inhibit the expression of IFN-λI , but the specific regulatory mechanism is still not clear.