肠道病毒71型感染人神经母细胞瘤细胞（SH-SY5Y）的miRNA表达谱

miRNA expression profiling of EV71 infected human neuroblastoma cells

目的 研究肠道病毒71型（EV71）感染神经细胞的miRNA表达谱,探讨miRNA在病毒感染神经细胞中的可能作用.方法 建立EV71感染人神经母细胞瘤细胞（SH-SY5Y）模型,收集感染后48 h细胞.以Taqman低密度芯片检测miRNA表达谱,使用实时RT-PCR对芯片结果进行验证并在TargetScan和miRanda网站预测靶基因,采用GO和KEGG分析靶基因功能.结果 成功建立EV71感染SH-SY5Y细胞模型,通过低密度芯片筛选出215种显著升高的miRNA和25种显著下调的miRNA.经过RT-PCR验证,3种miRNA（MiR-10a＊、miR-15b＊和miR-195）显著下调,7种miRNA（miR-10a、miR-342-5p、miR-483-5p、Let-7b、miR-99a、miR-140-5p和miR-21）显著上调,与芯片结果相符.GO分析显示发展进程和信号调节条目最富集靶基因.KEGG路径分析显示靶基因在肿瘤路径、蛋白水解、Wnt信号传导、黑素形成、粘附连接、MAPK信号通道最富集.结论 EV71感染神经细胞48 h后miRNA表达谱发生改变,10种变化的miRNA靶基因预测在发展进程、信号传导及凋亡中起着重要的作用,可为后期机制研究提供参考.

Objective To study the miRNA expression profiling in EV71 infected human neuroblastoma （SH-SY5Y） cells and to identify the possible effects of host miRNAs in the neurological pathogenesis of EV71 infection. Methods The EV71 infected SH-SY5Y cell model was established and the cells were collected after 48 h of infection. miRNA expression profiling in EV71 infected SH-SY5Y cells was performed using Taqman Low-Density Array （TLDA） and was validated using real-time RT-PCR. The putative target genes for the 10 candidate miRNAs were predicted using Targetscan and miRanda online algorithms. The target genes were then analyzed by GO enrichment and KEGG pathway analysis. Results The EV71 infected cell model was successfully established. The TLDA results showed 215 up-regulated and 25 down-regulated miRNAs in the EV71 infected cells compared with controls. The expression levels of 3 down-regulated miRNAs （miR-10a＊, miR-15b＊and miR-195） and 7 up-regulated miRNAs （miR-10a, miR-342-5p, miR-483-5p, Let-7b, miR-99a, miR-140-5p and miR-21） were confirmed by RT-qPCR. The verification results of 10 candidate miRNAs were basically in agreement with the array results. The GO enrichment analysis showed that the target genes were enriched into the category of developmental process and regulation of signaling. KEGG pathway analysis suggested that these target genes were involved in many important pathways, including pathways in cancer, endocytosis, Wnt signaling pathway, melanogenesis, adherens junction and MAPK signaling pathway. Conclusions The miRNA expression profiling of SH-SY5Y cells changed after 48h infected with EV71. The 10 miRNAs played an important role in developmental process, regulation of signaling and apoptosis. Our finding may assist future researches on the pathogenesis mechanisms of EV71 infection.