环状非编码RNA hsacirc0001613影响寨卡病毒复制的初步探究  

Effect of hsairc001613 on Zika virus replication:Preliminary study

作　　者：谢鹤 康澜 朱娅 代京宏 李世林[1] 李玉佳[1] 孙红刚 陈利民[1,2] 李彬 段晓琼 XIE He;KANG Lan;ZHU Ya;DAI Jing-hong;LI Shilin;LI Yujia;SUN Honggang;CHEN Limin;LI Bin;DUAN Xiaoqiong(Institute of Blood Transfusion,Chinese Academy of Medical Sciences and Peking Union Medical College,Chengdu 610052,China;Joint Laboratory for Transfusion Transmitted Diseases between Institute of Blood Transfusion,Chinese Academy of Medical Sciences and Nanning Blood Center)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]中国医学科学院输血研究所-南宁中心血站输血传播疾病联合实验室

出　　处：《中国输血杂志》2021年第5期472-476,共5页Chinese Journal of Blood Transfusion

基　　金：国家重点研发计划政府间国际科技合作重点专项(2018YFE0107500);科技部常规援外项目(KY201904011);四川省国际科技创新合作/港澳台科技创新合作项目(2020YFH0070)部分经费资助。

摘　　要：目的探究可经血液传播的寨卡病毒(Zika virus,ZIKV)体外感染细胞后对环状非编码RNA(hsa_circ_0001613)表达的影响及hsa_circ_0001613对寨卡病毒复制的影响。方法在12孔板每孔接种1.8×10^(5)个人类肺泡基底上皮细胞(A549)后24 h,用ZIKV GZ01感染A549细胞(MOI=0.5),感染后1~5 d每天分别收集细胞提取细胞内总RNA,通过荧光定量PCR(qRT-PCR)检测hsa_circ_0001613的相对表达量变化;在A549细胞中转染10nM siRNA-hsa_circ_0001613,特异性敲低hsa_circ_0001613的表达。转染后24 h用ZIKV GZ01感染细胞(MOI=0.05),然后分别在感染后1~5 d后提取细胞内RNA,感染后在3d收取蛋白,通过qRT-PCR和蛋白印迹法(western blot)检测ZIKV的复制水平及宿主抗病毒相关基因表达水平。此外通过双荧光报告基因试验检测干扰素刺激反应元件(ISRE)的活性。结果与未感染ZIKV的A549细胞相比,ZIKV感染A549细胞1~5 d后,hsa_circ_0001613相对表达降低。与转染siRNA阴性对照相比,在A549细胞中敲低hsa_circ_0001613表达后,ZIKV复制被显著抑制;且敲低hsa_circ_0001613表达水平后,IFN-α/β及一些干扰素刺激基因的表达明显升高,IFN-β诱导的ISRE活性显著增强。结论ZIKV感染A549细胞后,hsa_circ_0001613表达水平显著降低;特异性敲低hsa_circ_0001613表达水平后,明显抑制ZIKV复制。初步探索其机制是通过刺激宿主细胞中Ⅰ型干扰素、提高ISRE的活性及一些具有抗病毒活性的干扰素刺激基因的表达来抑制ZIKV复制。Objective To investigate the effect of transfusion-transmitted Zika virus(ZIKV)on the expression of non-coding circular RNA(hsa_circ_0001613)and the role of hsa_circ_0001613 in Zika virus replication.Methods Human adenocarcinomic alveolar basal epithelial cells(A549)were seeded on a 12-well plate at 1.8×10^(5)/well and infected with ZIKV at 0.05 MOI.The Total RNAs were isolated every day for 5 days after infection,and the relative expression level of hsa_circ_0001613 was detected by qRT-PCR.In addition,10 nM siRNA-hsa_circ_0001613 was transfected into 2×10^(5)/well A549 cells to specifically knock down the expression level of hsa_circ_0001613.24 h later,the cells were infected with ZIKV(MOI=0.05).Total RNAs were isolated at day 1-5 post-infection,proteins were extracted 96 h post-infection.ZIKV replication,relative host antiviral gene expression,and interferon stimulated response element(ISRE)activity were tested using qRT-PCR,western blot and dual luciferase assay,respectively.Results The relative expression of hsa_circ_0001613 decreased significantly after 1-5 days of ZIKV infection.Knockdown of hsa_circ_0001613 inhibited ZIKV replication.Meanwhile,hsa_circ_0001613 knockdown significantly upregulated IFN-α/βand its downstream interferon-stimulated genes(ISGs)expression,also increased ISRE activity.Conclusion ZIKV infection significantly suppressed hsa_circ_0001613 expression in A4549 cells.Preliminary study indicated that hsa_circ_0001613 knockdown inhibited ZIKV replication possibly through activating type-ⅠIFN signaling pathway as showed by increased ISGs expression and ISRE activity.

关 键 词：环状非编码RNA hsacirc0001613 寨卡病毒 病毒复制 Ⅰ型干扰素 

分 类 号：R373.9[医药卫生—病原生物学] R457[医药卫生—基础医学]