miR432*调控柯萨奇病毒A16型在人横纹肌肉瘤细胞中的复制

miR432* regulate the replication of Coxsackievirus A16 in rhabdomyosarcoma cells

摘要:

摘要:【目的】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。作者研究了miRNAs对柯萨奇病毒A16型(Coxsackievirus A16，CA16)在宿主细胞内复制的影响。【方法】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。通过实验，发现CA16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，预测可能作用于5'-UTR基因片段的miRNAs，检测miRNAs 对5'-UTR基因片段的作用。为了研究miRNAs分子对5'-UTR基因的调控作用是否可以体现在CA16病毒的复制过程中，在人横纹肌肉瘤(Rhabdomyosarcoma，RD)细胞中转染miRNAs mimics和inhibitors，利用Western blot和real-time PCR实验检验CA16病毒的复制和表达情况。【结果】实验结果表明，miR432*可以促进病毒在RD细胞中的复制和表达。反之，miR432* inhibitor有抑制病毒复制的作用。【结论】细胞内miR432*可以调控CA16在宿主细胞中的复制过程，本研究首次报导miR432*在CA16病毒复制过程中的调控作用。研究CA16病毒与宿主miRNAs的相互作用机制为进一步阐明CA16病毒感染与复制机理奠定了基础。

关键词:关键词:miRNAs，柯萨奇病毒A16型，人横纹肌肉瘤细胞，病毒复制

Abstract:

Abstract:［Objective］MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells．In this study，we examined miＲNAs’effects on the replication of Coxsackievirus A16 ( CA16) in rhabdomyosarcoma cells.［Methods］We constructed target gene of miRNAs screening system． We used 3' untranslated region (UTR) dual luciferase reporter analysis to identify putative miRNA targets in the CA16 virus genome．First，12 segments of CA16 virus genome 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，using online analysis programs we screened the miRNAs that may target to 5'-UTR．Furthermore，Western blot and real-time PCR test were used to study the effect of miRNAs on viral replication.［Results］The study showed that miR432* could stimulate the replication of CA16 virus．On the contrary，miR432* inhibitor could suppress CA16 virus replication.［Conclusion ］Cellular miRNAs could regulate the replication of CA16 virus in host cells．Our findings support the notion that the cellular miRNAs play an important role in the host and virus infection．

Key words:Keywords: miRNAs，Coxsackievirus A16，rhabdomyosarcoma cells，virus replication