模拟失重下氧化应激对EA.hy926细胞miRNA表达谱的影响及生物信息学分析

Effects of Oxidative Stress on the MicroRNAs Profiles of EA.hy926 Cells and Bioinformatics Analysis Under Simulated Microgravity

目的探索模拟失重条件下EA.hy926细胞氧化应激后microRNAs(miRNAs)表达谱的变化及生物学意义。方法 EA.hy926细胞分为对照组(Con组)、对照氧化应激组(Con+H2O2)、模拟失重组(MG组)和模拟失重后氧化应激组(MG+H2O2),采用Illumina HiSeq 2500平台开展miRNAs测序并筛选差异表达miRNAs。筛选出的miRNAs以qRT-PCR进行验证,对验证具有显著差异表达的miRNAs进行靶基因预测、靶基因Gene Ontology(GO)功能富集分析、KEGG信号通路分析以及STRING蛋白互作分析。结果模拟失重下氧化应激导致EA.hy926细胞内miRNAs表达谱发生改变。qRT-PCR结果显示,与Con+H2O2组相比,MG+H2O2组细胞内hsa-miR-29b-3p、hsa-miR-200c-3p表达增加,与miRNA测序结果一致。GO及KEGG通路显著性富集分析结果显示,hsa-miR-29b-3p靶基因显著富集于细胞外基质等生物学过程及局部黏附信号通路,hsa-miR-200c-3p靶基因显著富集于调控细胞代谢过程等生物学过程及miRNA癌症通路等信号通路。蛋白互作分析结果表明,hsa-miR-29b-3p靶基因中COL家族蛋白处于互作关键位置,hsa-miR-200c-3p靶基因中RHOA处于互作关键位置。结论模拟失重下氧化应激可导致EA.hy926细胞miRNAs表达出现显著差异,其中hsa-miR-29b-3p和hsa-miR-200c-3p可通过对靶基因及其信号通路调节对内皮细胞功能发挥调控作用。

Objective To explore the differentially expressed microRNAs(miRNAs)profiles and their biological significance induced by oxidative stress in EA.hy926 cells under simulated microgravity induced by clinostat.Methods EA.hy926 cells were divided into the control group(Con),oxidative stress for control group(Con+H2 O2),simulated microgravity group(MG)and oxidative stress for simulated microgravity group(MG+H2 O2).The Illumina HiSeq 2500 platform sequencing technology was used to perform miRNA sequencing(miRNA-seq)and screen differentially expressed miRNAs.The selected differentially expressed miRNAs were verified by qRT-PCR technology and their predicted target genes were analyzed with Gene Ontology(GO)enrichment,KEGG pathway and STRING protein interaction analysis by online analysis tools.Results The miRNA profiles changed in EA.hy926 cells induced by oxidative stress under simulated microgravity.qRT-PCR showed the expression of hsa-miR-29 b-3 p and hsa-miR-200 c-3 p increased in MG+H2 O2 group compared with Con+H2 O2 group,which were consistent with the results of miRNA-seq.GO and KEGG pathway significant enrichment analysis showed that the target genes of hsa-miR-29 b-3 p were significantly enriched in the biological process of extracellular matrix organization and focal adhesion pathway,etc.Target genes of hsa-miR-200 c-3 p were significantly enriched in the biological process of regulation of cellular metabolic process and miRNA in cancer pathway,etc.Protein-protein interaction analysis results showed that proteins of COL family were in the key position of hsa-miR-29 b-3 p target genes,and RHOA protein was in the key position of hsa-miR-200 c-3 p target genes.Conclusion Oxidative stress under simulated microgravity induces significantly different expression of miRNAs in EA.hy926 cells.Hsa-miR-29 b-3 p and hsa-miR-200 c-3 p may participate in the dysfunction regulation of endothelial cells under microgravity by regulating target genes and pathways.