小干扰RNA靶向5'非编码区抗肠道病毒71型感染的研究

摘要

Objective To screen and identify effective small interfering RNA (siRNAs) targeting the conserved 5’ untrans-lated region (UTR) of the enterovirus 71 (EV71) genome. Methods Double-stranded siRNAs were designed to target the 5’UTR of the EV71 genome. The cytotoxicity effect of siRNAs on rhabdomyosarcoma (RD) cells was evaluated. The cytopathic effect on EV71-infected RD cells was observed under phase-contrast microscopy and the effective siRNAs were screened out by cell viability assay and real-time TaqMan RT-PCR assay. Results All the siRNAs did not exhibit any cytotoxic effects on the viability and growth of RD cells. Transfection of si-1 and si-2 targeting the 5’ UTR in EV71 genome into RD cells signiifcantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability and reduced the EV71 RNA transcripts. And the inhibitory effect of si-1/si-2 on EV71 replication was sequence-speciifc. Conclusions The 2 siRNAs (si-1 and si-2) targeting the conserved 5’UTR of the EV71 genome may have broad spectrum antiviral effects on many epidemic EV71 strains.%目的：筛选靶向肠道病毒71型（EV71）基因组保守的5’非编码区（UTR）的有效小干扰RNA（siRNAs），确定有效靶点序列。方法设计合成靶向EV71基因组5’UTR的siRNAs；细胞毒性实验检测siRNAs对RD细胞的生存和生长的影响，显微镜观察siRNAs对EV71致细胞病变效应的影响，细胞生存实验、TaqMan实时定量RT-PCR实验筛选有效siRNAs。结果 siRNAs未影响RD细胞的生存及生长，对培养细胞无明显毒性。靶向EV71基因组5’UTR的si-1和si-2抗病毒效能最显著，能够延缓及减轻EV71感染致细胞病变效应，明显提高感染细胞的生存率，降低感染细胞中EV71 RNA的转录水平，且这种作用具有序列特异性。结论筛选出的靶向EV71基因组保守5’UTR的2个有效siRNAs（si-1和si-2），对多株EV71中国流行株可能具有广谱抗病毒效能。