miR373和miR542-5p调控肠道病毒71型在人横纹肌肉瘤细胞中的复制

miR373 and miR542-5p regulate the replication of Enterovirus 71 in rhabdomyosarcoma cells

摘要:

研究发现microRNAs (miRNAs) 可以参与调控病毒在宿主细胞内感染和复制的过程。为了揭示miRNAs是否参与肠道病毒71型 (Enterovirus 71, EV71) 的感染与复制，研究了miRNAs 对EV71病毒在宿主细胞内复制的影响。构建miRNAs靶基因筛选系统，在双荧光素酶报告体系的pMIR载体插入病毒基因，如果插入的基因序列能被细胞内的miRNAs靶向调控，报告基因的表达将发生变化。实验发现EV71病毒5′-UTR基因可能是miRNAs的作用靶标。随后利用miRNAs在线分析软件预测并验证可能作用于5′-UTR基因片段的miRNAs。为了研究miRNAs分子对5′-UTR基因的调控作用是否可以体现在EV71病毒的复制过程中，在人横纹肌肉瘤(Rhabdomyosarcoma, RD)细胞中转染miRNAs mimics，利用Western blotting和real-time PCR实验检验EV71病毒的复制和表达情况。实验结果表明，miR373和miR542-5p可以通过作用于EV71病毒5′-UTR基因从而抑制病毒在RD细胞中的复制和表达。细胞内miR373和miR542-5p可以调控EV71在宿主细胞中的复制过程。研究EV71病毒与宿主miRNAs的相互作用机制为进一步阐明EV71病毒感染与复制机理奠定了基础。

Abstract:

MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we examined miRNAs effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of miRNAs screening system. 3′untranslated region (UTR) dual luciferase reporter analysis was used to identify putative miRNA targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5′-UTR was significantly downregulated. Then we screened the miRNAs that may target to 5′-UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. The study showed that miR373 and miR542-5p could suppress the replication of EV71 virus through binding to the 5′-UTR gene. Cellular miRNAs could regulate the replication of EV71 virus in host cells, and our paper should report the role of miR373 and miR542-5p in this regulation for the first time. Our findings supported the notion that the cellular miRNAs might be essential in the host-pathogen interactions.