小鼠骨髓间充质干细胞衰老相关mRNA-miRNA分析

Analysis of mRNA-miRNA associated with aging mouse bone marrow mesenchymal stem cells

目的 骨髓间充质干细胞(BMSC,Bone marrow mesenchymal stem cells)是来源于骨髓腔并具有多项分化潜能的干细胞。衰老损害了骨髓间充质干细胞的多向分化特性,导致与年龄相关的骨质流失,其关键调控因子仍不清楚。方法从Gene Expression Omnibus数据库下载mRNA(GSE44403)和microRNA(GSE57127)的表达数据集。R软件鉴定差异表达基因(DEG,Differentially expressed genes)和microRNAs(DEM,Differentially expressed microRNAs)。使用MiRTarbase预测DEM的靶点,并将DEM的靶基因与DEG取交集。用David数据库对获得的重叠基因进行功能富集化分析。利用Cytoscape进行PPI网络构建、Hub基因和重要模块的鉴定以及mRNA-miRNA调控网络的可视化。构建体外衰老细胞模型,RT-qPCR验证筛选的关键基因及相关microRNA。结果 在年轻和老年小鼠之间共鉴定出1917个差异mRNA和197差异microRNA。选择了miR-690、miR-494-3p、miR-181a、miR-142-5p、miR-188和miR-34b等前20个调控异常的DEM作为关键microRNAs。DEG与DEM靶点的重叠基因主要集中在PI3K-Akt信号通路、ErbB信号通路和EGFR信号通路中。在构建的PPI网络中,筛选出TOP10中的Hub基因(包括mTOR、Insr、GSK3b和Ppp2ca)和顶端模块。通过miRNA-mRNA调控网络及RT-qPCR验证结果,确定了miR-34b-5p-Insr重要调控对。结论 本研究发现衰老小鼠的间充质干细胞中存在miR-34b-5p-Insr重要调控对,可成为调控衰老BMSC细胞功能的潜在靶点。

Objective Bone marrow mesenchymal stem cells(BMSC) are stem cells derived from the bone marrow cavity with multiple differentiation potentials.However,aging impairs the regenerative properties of BMSC and the molecular mechanisms under which remain elusive.Methods The mRNA(GSE44403) and microRNA(GSE57127) expression datasets were downloaded from the Gene Expression Omnibus dataset.Differentially expressed genes(DEGs) and microRNAs(DEMs)were identified by R software.MiRTarbase was used to predict targets of DEMs.Then functional enrichment analysis was performed by DAVID using the interaction of DEGs and DEM targets.Cytoscape was used for PPI network construction,Hub gene and important modules identification and mRNA-miRNA regulatory network visualization.Finally,the Hub genes and interrelate microRNAs were verified by RT-qPCR using the senescent cell model in vitro.Results 1917 significant DEGs and 197 DEMs were identified between young and old mice.Top 20 dysregulated DEMs including miR-690,miR-494-3p,miR-181a,miR-142-5p,miR-188 and miR-34b were selected as key microRNAs.The overlapped genes between DEGs and DEM targets were mainly enriched in PI3K-Akt signaling pathway,ErbB signaling pathway and EGFR signaling pathway.A PPI network was constructed and top10 Hub genes(including mTOR,Insr,GSK3b and Ppp2ca) and the top module was selected.The important regulatory pair miR-34b-5p-Insr was identified by miRNA-mRNA regulatory network and RT-qPCR verification results.Conclusions Our study concluded that the important regulatory pair miR-34b-5p-Insr exists in mesenchymal stem cells of aging mice,which can be potential targets for regulating the function of aging BMSC.