摘要
目的探讨丙型肝炎病毒(HCV)调控ADAR1逃避先天免疫的潜在机制。方法通过siRNA敲低ADAR1(敲低组)或GFP(对照组)后,检测炎症因子和干扰素的mRNA水平、IFNγ的分泌水平和HCV的复制水平。HCV感染Huh7细胞(HCV组)或不感染Huh7细胞(MOCK组)后,检测ADAR1的mRNA水平和蛋白水平,以及ADAR1的启动子活性。通过miRDB在线预测ADAR1的miRNA。HCV感染Huh7细胞后,检测上述miRNA的表达水平。过表达差异miRNA或negative control(对照组)后检测ADAR1的表达水平。结果与对照组比较,敲低组炎症因子和干扰素的mRNA水平均上升,IFNγ的分泌水平上升,HCV的复制水平下降(P<0.05)。与MOCK组比较,HCV组ADAR1在mRNA水平和蛋白水平均明显上升(P<0.05),但是,ADAR1的启动子活性没有明显变化(P>0.05)。miRDB在线分析发现有多种潜在靶向ADAR1的miRNA。HCV感染Huh7细胞后,通过实时荧光定量PCR检测上述miRNA,与对照组比较,hsa-miR-613、hsa-miR-206、hsa-miR-135b-5p、hsa-miR-135a-5p、hsa-miR-6803-5p、hsa-miR-4530、hsa-miR-3915的表达水平均下降,差异有统计学意义(P<0.05)。过表达上述miRNA后,与对照组比较,hsa-miR-135b-5p和hsa-miR-135a-5p能够在mRNA水平减少ADAR1的表达(P<0.05)。与对照组比较,过表达hsa-miR-135a-5p或miR-135b-5p后,ADAR1的表达水平均下降(P<0.05)、IFNγ的分泌水平上升(P<0.05)、HCV的复制水平下降(P<0.05);与对照组比较,敲低hsa-miR-135a-5p或miR-135b-5p后,ADAR1的表达水平均上升,IFNγ的分泌水平下降,HCV的复制水平上升(P<0.05)。结论HCV感染后,Huh7细胞中hsa-miR-135a-5p和miR-135b-5p的表达水平下降,其靶向ADAR1 mRNA的3′端非编码区的水平下降,随后ADAR1的表达水平上升,抑制了宿主的先天免疫,促进了HCV的复制。
Objective To investigate the potential mechanism of hepatitis C virus(HCV)regulation of ADAR1 to evade innate immunity.Methods The mRNA levels of inflammatory factors and interferons,the secretion level of IFNγand the replication level of HCV were detected after knockdown of ADAR1 by siRNA(knockdown group)or GFP(control group).mRNA levels and protein levels of ADAR1 were detected after HCV infection of Huh7 cells(HCV group)or not(MOCK group),as well as promoter activity of ADAR1.The miRNA of ADAR1 was predicted online by miRDB.After HCV infection of Huh7 cells,the expression levels of the above miRNAs were detected.The expression levels of ADAR1 were detected after overexpression of differential miRNAs or negative control(control).Results Compared with control group,knockdown group showed increased expression of mRNA levels of both inflammatory factors and interferons,increased secretion levels of IFNγ,and decreased replication levels of HCV(P<0.05).Compared with MOCK group,ADAR1 increased significantly in HCV group at both mRNA level and protein level(P<0.05).However,there was no significant change in the promoter activity of ADAR1(P>0.05).miRDB online analysis identified a variety of miRNAs that potentially target ADAR1.hsa-miR-613,hsa-miR-206,hsa-miR-135b-5p,hsa-miR-135a-5p,hsa-miR-6803-5p,hsa-miR-4530,hsa-miR-3915 expression levels decreased with statistically significant differences(P<0.05).After overexpression of the above miRNAs,hsa-miR-135b-5p and hsa-miR-135a-5p were able to reduce the expression of ADAR1 at the mRNA level compared with control group(P<0.05).Compared with control group,the expression level of ADAR1 decreased,the secretion level of IFNγincreased,and the replication level of HCV decreased(P<0.05)after overexpression of either hsa-miR-135a-5p or miR-135b-5p.Compared with control group,knockdown of hsa-miR-135a-5p or miR 135b-5p,the expression level of ADAR1 increased,the secretion level of IFNγdecreased,and the replication level of HCV increased(P<0.05).Conclusion After HCV infection,the expression levels of hsa-miR-135a-5p and miR-135b-5p in Huh7 cells,which target the 3′-terminal non-coding region of ADAR1 mRNA decreased,followed by an increase in the expression level of ADAR1,suppressed host innate immunity and promoted HCV replication.
作者
王本鑫
王福义
孟凡鑫
WANG Benxin;WANG Fuyi;MENG Fanxin(Department of Clinical Laboratory Medicine,Affiliated Central Hospital of Shenyang Medical College,Shenyang,Liaoning 110024,China)
出处
《国际检验医学杂志》
CAS
2021年第10期1213-1218,共6页
International Journal of Laboratory Medicine