RNA编辑酶ADAR1在Huh7.5.1细胞中抑制HCV复制的研究

ADAR1 mediated-inhibition of HCV replication in Huh7.5.1 cells

目的:探究干扰素在Huh7.5.1细胞中对RNA编辑酶(adenosine deaminases acting on RNA 1,ADAR1)表达的影响,以及ADAR1对丙型肝炎病毒(hepatitis C virus,HCV)复制的调控作用。方法:干扰素(IFN-α,30 IU/m L)刺激Huh7.5.1细胞后,利用real-time PCR和Western blot检测ADAR1表达水平的变化,同时HCV以一定滴度(0.2 MOI)感染Huh7.5.1细胞,观察IFN-α对HCV复制的影响;利用si RNA降低ADAR1表达,过表达质粒(ADAR1 p110和p150)增加ADAR1表达,并进一步利用基因编辑技术建立ADAR1稳定敲除细胞株,明确ADAR1与HCV的复制关系。结果:IFN-α诱导Huh7.5.1细胞ADAR1 p150表达(48 h:t=10.400,P=0.007;72 h:t=19.390,P=0.002),而不影响ADAR1 p110表达(48 h:t=0.806,P=0.472;72 h:t=1.929,P=0.133),同时IFN-α可显著抑制HCV复制(48 h:t=10.170,P=0.001;72 h:t=35.810,P=0.000)。ADAR1 p110和p150过表达48 h后,HCV病毒核酸和蛋白水平均明显低于空白对照(P=0.004,P=0.015);ADAR1 si RNA干扰后,HCV复制增加了2倍以上,差异显著(t=13.530,P=0.000);利用Crispr-cas9技术敲除掉Huh7.5.1中的ADAR1后,HCV复制也明显增加(t=5.259,P=0.023);ADAR1干扰后IFN-α仍然存在对HCV明显的抑制作用(t=9.146,P=0.001)。结论:IFN诱导蛋白ADAR1可以抑制HCV复制,且ADAR1部分介导了IFN-α对HCV复制的抑制。

Objective：To explore the effect of interferon（IFN-α）on expression of adenosine deaminases acting on RNA 1（ADAR1）in Huh7.5.1 cells and the regulation of ADAR1 on HCV replication. Methods：The expressions of ADAR1 were measured in Huh7.5.1 cells post-treatment by IFN-α（30 IU/m L）,and the effects of IFN-α modulation were determined through addition of IFN-α following addition of infectious JFH HCV（0.2 MOI）to Huh7.5.1 cells. ADAR1 si RNA knockdown,overexpression and knockout in Huh7.5.1 cells were incorporated to testify the relation of ADAR1 and HCV replication. Results：IFN-α induced the significant expression of ADAR1 p150 on both RNA and protein level（48 h：t=10.400,P=0.007;72 h：t=19.390,P=0.002）,while the level of ADAR1 p110 remain stable（48 h：t=0.806,P=0.472;72 h：t=1.929,P=0.133）,and the level of HCV RNA decreased significantly（48 h：t=10.170,P=0.001;72 h：t=35.810,P=0.000）. Overexpression of ADAR1 p110 and p150 inhibited HCV replication on the level of RNA and protein（P =0.004,P =0.015）. The replication of HCV increased by two folds after ADAR1 si RNA knockdown（t =13.530,P =0.000）.Compared to intact cells,ADAR1 knockout Huh7.5.1 cells enhanced HCV replication（t=5.259,P=0.023）. IFN-α still showed significant inhibitory effect even after ADAR1 knockdown by si RNA（t =9.146,P =0.001）. Conclusion：ADAR1 induced by IFN-α inhibits HCV replication in Huh7.5.1 cells,and ADAR1 may partially mediate the inhibitory effect of IFN-α on HCV replication.