骨质疏松hub基因筛选验证及互作miRNA预测

The analysis and verification of osteoporosis associated hub genes and prediction of miRNA interactions

目的筛选老年性骨质疏松症hub基因及其互作的miRNA。方法从GEO数据库下载GSE35956基因芯片数据集,利用R语言筛选骨质疏松症患者骨髓间充质干细胞中的差异表达基因(Degs)。利用DAVID数据库对Degs进行GO富集和KEGG通路分析。采用String数据库和Cytoscape软件分析蛋白互作网络,筛选hub基因。通过Targetscan数据库预测与hub基因存在互作关系的miRNA,并通过funrich数据库对预测出的miRNA进行富集分析。提取并培养3~4月龄(青年组)和18~20月龄(老年组)C57BL/6小鼠的骨髓间充质干细胞,采用荧光定量PCR(qPCR)验证2组小鼠的骨髓间充质干细胞中hub基因表达情况。结果骨质疏松症患者骨髓间充质干细胞中共鉴定出982个上调的Degs和99个下调的Degs。Degs主要富集到质膜黏附分子介导的同型细胞黏附和钙离子结合等生物过程,主要参与了神经活性配体-受体相互作用信号通路。PPI互作网络分析筛选出8个核心基因:瘦素(LEP)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、WT1转录因子(WT1)、SRY-Box转录因子10(SOX10)、整合素β2(ITGB2)、白蛋白(ALB)、胆囊收缩素(CCK)和骨形态发生蛋白7(BMP7)。Targetscan预测了105个miRNA和其中6个核心基因存在互作关系。qPCR验证结果表明,老年组小鼠骨髓间充质干细胞中BMP7、CCK、ITGB2、SOX10基因表达水平较青年组上调,与生物信息学分析结果相一致。结论筛选出的核心基因表达水平与老年小鼠骨髓间充质干细胞分化程度相关,鉴定得到的miRNA可能成为老年性骨质疏松症诊断标志物及治疗靶点。

Objective To screen hub genes and miRNA interaction in aging osteoporosis.Methods The GSE35956 gene chip data set was downloaded from GEO database,and the differentially expressed genes(Degs)in bone marrow mesenchymal stem cells were screened by R language in patients with osteoporosis.GO enrichment and KEGG pathway were analyzed in Degs using DAVID database.String database and Cytoscape software were used to analyze the protein interaction network and screen hub genes.The mirnas interacting with hub genes were predicted by Targetscan database,and the predicted mirnas were enriched by funrich database.Bone marrow mesenchymal stem cells from 3 to 4 month-old(the young group)and 18 to 20 month-old(the old group)in C57BL/6 mice were extracted and cultured.The expression levels of miRNA in bone marrow mesenchymal stem cells were verified by qPCR technique in the young and the old groups.Results A total of 982 up-regulated Degs and 99 down-regulated degs were identified in patients with osteoporosis.Degs were mainly enriched in plasma membrane adhesion molecule-mediated homologous cell adhesion and calcium ion binding,and were mainly involved in neuroactive ligand-receptor interaction signaling pathways.PPI interaction network analysis identified eight core genes:leptin(LEP),lymphocyte-specific protein tyrosine kinase(LCK),WT1 transcription factor(WT1),SRY-box transcription factor 10(SOX10),integrin subunit beta 2(ITGB2),albumin(ALB),cholecystokinin(CCK)and bone morphogenetic protein 7(BMP7).The interaction between 105 miRNA and 6 core genes was predicted by Trargetscan.qPCR results showed that BMP7,CCK,ITGB2 and SOX10 genes in bone marrow mesenchymal stem cells were significantly upregulated in the old group compared with those of the young group.It is consistent with the results of bioinformatics analysis.Conclusion The expression levels of the selected core genes are related to the differentiation degree of bone marrow mesenchymal stem cells in aged mice.The identified miRNA may be a diagnostic marker and a therapeutic target for aging osteoporosis.