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

摘要:

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/mL）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siRNA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 siRNA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。

Abstract:

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/mL），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 siRNA 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 re－main 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 siRNA 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 siRNA（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.