柯萨奇病毒B3型感染引起HeLa细胞内源性小干扰RNA的变化  

The change of endogenous siRNAs in coxsackie virus type B3-infected HeLa cells

作　　者：姚海兰[1] 宋娟[2] 王欣玲[2] 王瑞芳 宋芹芹[2] 史冰田 韩俊[2] YAO Hailan;SONG Juan;WANG Xinling;WANG Ruifang;SONG Qinqin;SHI Bintian;HAN Jun(Department of Biochemistry&Immunology,Capital Institute of Pediatric,Beijing 100020,China;State Key Laboratory of Infectious Disease Prevention and Control,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]首都儿科研究所生物化学与免疫室,北京100020 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《微生物与感染》2020年第2期98-103,共6页Journal of Microbes and Infections

基　　金：重大传染病防治国家科技重大专项(2018ZX10102001、2018ZX10711001、2018ZX10734404);传染病预防控制国家重点实验室发展基金项目(2011SKLID104)。

摘　　要：探究柯萨奇病毒B3型(Coxsackie virus type B3,CVB3)感染的细胞是否诱导内源性小干扰RNA(small interfering RNA,siRNA)的产生。以CVB3接种HeLa细胞,在细胞培养箱(5%CO 2、37℃)孵育1 h。随后加入含2%血清的细胞维持液,继续培养3 h和6 h后收集细胞。用高通量测序(二代测序)试剂盒提取细胞RNA,并反转录合成cDNA,构建文库,上机测序。过滤数据,去除插入片段过长的序列、低质量序列、poly A序列和小片段序列,与已知的小RNA数据库比对鉴定siRNA。通过茎-环反转录-聚合酶链反应,证实内源性siRNA在感染CVB3后的表达。结果显示,感染CVB33 h和6 h后,HeLa细胞产生了多种内源性siRNA。其中内源性siRNA(novel_sir3502和novel_sir2806)在感染后3 h和6 h均可持续表达。经比对,novel_sir3502和novel_sir2806均可以识别45S和28S核糖体前体RNA。结果提示,CVB3感染可能干扰核糖体成熟。To explore whether coxsackie virus type B3(CVB3)infection can induce the expression of endogenous siRNA,siRNAs were analyzed in CVB3-infected HeLa cells at 3h and 6h post-infection(pi)by the high-throughput sequencing.The results showed that CVB3 induced 14 endogenous siRNA and 5 endogenous siRNA in HeLa cells at 3 h and 6 h pi,respectively.Two sustainably expressed endogenous siRNAs were further confirmed at 3 h and 6 h pi using quantitative PCR.Results of sequence homology alignment showed that two siRNAs could recognize the precursor RNA of 45S ribosome and 28S ribosome.The results suggest that CVB3 infection may interfere ribosome maturation.

关 键 词：柯萨奇病毒B3型 HELA细胞 内源性小干扰RNA 

分 类 号：R373[医药卫生—病原生物学] R34[医药卫生—基础医学]