高通量测序确定肠道病毒A71型感染致宿主细胞miRNA表达谱的改变

Determination of miRNA profile alteration of host cells induced by enterovirus-A71 through high-throughput sequencing

目的确定肠道病毒A71型(Enterovirus-A71,EV-A71)感染人扁桃体上皮细胞后miRNA表达谱的改变。方法EV-A71以感染复数为1感染人扁桃体上皮细胞6 h后,采用Trizol提取总RNA,构建小RNA文库,在Illumina NextSeq 500平台进行高通量测序,处理数据后确定差异表达的已知和新型miRNA种类及其靶基因,进行基因本体和信号途径分析;使用psRNATarget预测具有潜在结合EV-A71基因组的差异表达的miRNA种类。实时定量RT-PCR检测差异表达miRNA的变化倍数。结果通过高通量测序共鉴定61个已知差异表达的miRNA(下调21个、上调40个)和559个新型miRNA,新型miRNA具有典型的前miRNA的二级发卡结构。实时定量RT-PCR确定hsa-miR-517b-3p和hsa-miR-199a-5p的变化倍数与高通量测序结果基本一致。差异表达miRNA靶基因涉及到不同的生物学过程和信号途径。共鉴定出24个差异表达的miRNA(5个已知miRNA,19个新型miRNA)具有与EV-A71结合的潜在"种子序列"。结论EV-A71感染人扁桃体上皮细胞后引起miRNA表达谱的显著改变,且差异表达的miRNA可能靶向结合EV-A71基因组进而调控EV-A71复制。

Objective To determine the alteration of miRNA profile of human tonsillar epithelial cells induced by enterovirus-A71(EV-A71)infection.Methods Human tonsillar epithelial cells UT-SCC-60B were infected with EV-A71 at multiplicities of infection(MOI)of 1 and total RNA was extracted using Trizol reagent.Small RNA library was constructed and high-throughput sequencing was performed using Illumina NextSeq 500.Differential significantly expressed known and novel miRNAs and putative targets were selected after the processing of raw data.Gene ontology(GO),kyoto encyclopedia of genes and genomes(KEGG)pathways were analyzed through online database.Kinds of miRNA could target EV-A71 genome was determined through psRNATarget.Validations of random selected miRNAs were done through real-time RT-PCR.Results A total of 61 known significantly expressed miRNAs(21 miRNAs were down-regulated and 40 miRNAs were up-regulated)and 559 novel significantly expressed miRNAs were identified through high-throughput sequencing.Novel significantly expressed miRNA had typical"hairpin structure"of pre-miRNA.Fold changes of hsa-miR-517b-3p and hsa-miR-199a-5p which was determined by real-time RT-PCR had similar change trends with high-throughput sequencing.Putative targets of significantly expressed miRNA were referred to different biological processes and signaling pathways.A total of 24 significant miRNAs(5 known significantly expressed miRNAs and 19 novel significantly expressed miRNAs)had"seed sequence"in EV-A71 genome.Conclusions Expression of miRNA profile in UT-SCC-60B was significantly changed by EV-A71 infection and the identified significantly expressed miRNAs potential target EV-A71 genome to regulate EV-A71 replication.