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【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。
【Objective】 The study found that microRNAs (miRNAs) can participate in the regulation of virus infection and replication in host cells. The authors investigated the effect of miRNAs on Coxsackievirus A16 (CA16) replication in host cells. 【Method】 The miRNAs target gene screening system was constructed. The viral gene was inserted into the pMIR vector of dual luciferase reporter system. The expression of reporter gene will be changed if the inserted gene sequence can be regulated by miRNAs in cells. Through experiments, it was found that the CA16 virus 5’-UTR gene may be the target of miRNAs. Then miRNAs online analysis software was used to predict the miRNAs that might act on the 5’-UTR gene fragment and detect the effect of miRNAs on the 5’-UTR gene fragment. In order to investigate whether miRNAs modulate the 5’-UTR gene expression in CA16 virus replication, miRNAs mimics and inhibitors were transfected in human rhabdomyosarcoma (RD) cells. Western blot and real-time PCR Experiment to examine the replication and expression of CA16 virus. 【Result】 The experimental results show that miR432 * can promote the replication and expression of the virus in RD cells. In contrast, miR432 * inhibitor has the effect of inhibiting viral replication. 【Conclusion】 Intracellular miR432 * can regulate the process of CA16 replication in host cells. This study reports for the first time the role of miR432 * in the replication of CA16 virus. The study of the interaction mechanism between CA16 virus and host miRNAs laid the foundation for further elucidating the mechanism of CA16 virus infection and replication.