MicroRNA-155对3种可经输血传播RNA病毒复制的影响及机制探究

Role of MicroRNA-155 on the replication of three kinds of blood transfusion transmitted RNA virus and its mechanism

目的 了解经血传播的RNA病毒感染细胞后对小分子非编码RNA-155 (miR-155)的诱导作用及miR-155对这类病毒复制的影响。方法 在2×10~5/mL肝癌细胞系Huh7.5.1中分别感染3种RNA病毒HCV、DENV和ZIKV,感染复数(MOI)为0.2,感染后48 h,提取胞内RNA,通过荧光定量PCR (qRT-PCR)的方法,检测病毒感染后miR-155的相对表达变化;同时在2×10~5/mL Huh7.5.1细胞中转染10 pmol的miR-155模拟物(miR-155 mimic)使miR-155高表达,转染后12 h分别接种0.2 MOI的3种病毒,感染后48 h提取胞内RNA,通过荧光定量PCR检测3种病毒RNA的复制水平及宿主抗病毒先天免疫相关基因表达水平。结果 与未感染病毒的Huh7.5.1细胞相比,HCV、DENV及ZIKV感染Huh7.5.1细胞48 h后,miR-155的相对表达分别升高了132倍、157倍和8倍。与转染阴性对照组相比,miR-155高表达的Huh7.5.1细胞中,HCV、ZIKV和DENV的复制分别降低了48%、54%和70%;miR-155高表达的Huh7.5.1细胞中,IFNβ及一些干扰素刺激基因的表达明显升高。结论 HCV、DENV和ZIKV感染Huh7.5.1细胞后,显著诱导细胞中miR-155的表达;miR-155在细胞中高表达后,通过调节宿主细胞中Ⅰ型干扰素及一些干扰素刺激基因的表达来抑制HCV、DENV和ZIKV的复制。

Objective To investigate the induction of three kinds of blood transfusion transmitted RNA virus on miR-155 and the role of miR-155 in HCV,DENV and ZIKV replication.Methods Huh7.5.1 cells were seeded on a 12-well plate at 2×105/mL and infected with HCV,DENV and ZIKV at 0.2 MOI,respectively.The cell RNAs 48 hours after infection were harvested and the relative expression of miR-155 was tested using qRT-PCR.Meanwhile,Huh7.5.1 cells were seeded on a 12-well plate at 2×105/mL and transfected miR-155 mimic at 10 pmol/mL 12 h before virus infection,and RNA was harvested 48h post-infection and relative gene mRNA was tested using qRT-PCR.Results The relative expression of miR-155 increased significantly by 132 folds,157 folds and 8 folds in HCV,DENV and ZIKV infected Huh7.5.1 cells,respectively.And miR-155 overexpression inhibited HCV,ZIKV and DENV replication by about 48%,54%and 70%,respectively compared with the negative mimic transfection cells.MiR-155 overexpression upregulated IFNβand its downstream interferon stimulated genes(ISGs)expression.Conclusion HCV,DENV and ZIKV infection induce miR-155 expression in Huh7.5.1 cells;miR-155 inhibits HCV,DENV and ZIKV replication through regulating IFN pathway and ISGs expression.