| Objectives The purpose of this research is to find the differentially expressed genes(DEGs)between elderly osteoporotic patients and healthy people with bioinformatics analysis,understand the biological process and function of DEGs related to osteoporosis,and screen the core genes by bioinformatics analysis and experimental verification.Methods 1 The microarray data GSE35956 was downloaded from the GEO database to screen the DEGs between osteoporotic patients and healthy people through R language,and the DEGs obtained were subjected to GO enrichment analysis and KEGG pathway enrichment analysis through David bioinformatics resources 6.8 online database.The Protein-protein interaction(PPI)analysis was performed by STRING database,the hub genes were obtained by further analysis using Cytoscape cyto Hubba.2 The experimental verification was conducted as following processes.The mRNA of mouse bone marrow stromal cells from aged and young mice was extracted,and the expression of hub gene in aged and young mice was verified by q PCR.The femurs of aged and young mice were stained with HE and immunohistochemistry to compare the morphological difference.The expression of core genes in aged and young mice was evaluated by Western blot at the protein level.3 The miRNAs interacted with the hub genes were predicted by Cy TargetLinker,then,the enrichment analysis of miRNAs interacted with hub genes were performed by Fun Rich version 3.1.4.Results 1 A total of 982 upregulated DEGs and 99 downregulated DEGs were identified by the R language limma package analysis.2 GO enrichment analysis showed that upregulated DEGs were mainly located in the extracellular region,plasma membrane and cell vesicles,and regulated the functions of calcium binding,neuropeptide receptor activity,MHC class Ⅱ receptor activity,etc.Down-regulated differentially expressed genes are mainly located in the cytoplasm,regulate the function of PDZ binding domain,and act on the positive regulation of apoptosis and cartilage development.According to KEGG pathway enrichment analysis,up-regulated DEGs are mainly involved in neuroactive ligand-receptor interaction pathway,JAK-STAT signaling pathway and PI3K-AKT signaling pathway,while down-regulated DEGs are mainly involved in histidine metabolism pathway.3 PPI interaction network analysis showed that we obtained multiple protein modules.GO and KEGG enrichment analysis of the top three protein modules showed that protein module A was mainly located in the nucleus and other parts,regulating the function of protein binding,and participating in DNA repair and cell division;protein module B was mainly located in the extracellular region,the wall of endocytosis vesicles and the postsynaptic membrane.It regulates the functions of ionotropic glutamate receptor activity,extracellular glutamate-gated ion channel activity and MHC class Ⅱ receptor activity,and participates in the processes of T cell differentiation,proteolysis,zymogen activation and appetite regulation.Protein module C mainly regulates transcription factor activity,sequence-specific DNA binding and sequence-specific DNA binding functions of RNA polymerase Ⅱ core promoter.Involved in RNA polymerase Ⅱ promoter transcription,positive regulation of RNA polymerase Ⅱ promoter transcription and negative regulation of neuronal differentiation,KEGG pathway enrichment results show that protein module C mainly concentrated in Hippo signaling pathway.4 The top 15 core genes were screened by four algorithms of cyto Hubba plug-in,and eight core genes were obtained by superimposing the screening results of four algorithms: CCK,LEP,BMP7,LCK,WT1,SOX10,ITGB2 and ALB.5 The expression levels of BMP7,CCK,LEP and ITGB2 were increased in mesenchymal stem cells of aged and young adults by real-time PCR.Through HE staining,it was found that the number of bone trabeculae was significantly reduced in the old group compared with the young group,and the number of bone fossa in the dry end was significantly reduced.Immunohistochemical staining and Western blot was used to verify that BMP7,CCK,LEP and ITGB2 expression was increased in the bone marrow of the aged mice compared with the young mice.6 Cy Target Linker predicted that 105 miRNAs might interact with six of these core genes.The enrichment analysis of the obtained miRNAs showed that the enriched miRNAs were mainly present in the cytoplasm,perform signal transduction,cell communication and participate in transcription factor activity,protein serine /threonine kinase activity and other biological processes.KEGG pathway enrichment results show that miRNA is mainly involved in proteoglycan Syndecan-mediated signal transduction,TRAIL signaling pathway and Erb B receptor signaling network.Conclusions 1 Based on bioinformatics data analysis,we screened differentially expressed genes and key protein modules between elderly patients with osteoporosis and healthy subjects and performed GO and KEGG pathway enrichment analysis,which clarified the biological processes,cellular functions,and signaling pathways involved.CCK,LEP,BMP7,LCK,WT1,SOX10,ITGB2,and ALB were selected as the core differentially expressed genes.2 Randomly selected 4 of these 8 core genes for experimental validation and found differential expression of the CCK,LEP,BMP7,and ITGB2 genes similarly within the cells of bone tissue from aged versus young mice,which is in agreement with the results of bioinformatic prediction analysis and suggests that bioinformatic prediction analysis may provide new insights into osteoporosis related research in future.3Bioinformatics data analysis predicted that 105 miRNAs might interact with 6 of the 8 core genes,which mainly perform functions such as signal transduction,cell communication,and participate in biological processes such as transcription factor activity and protein serine/threonine kinase activity,which may provide some reference for subsequent osteoporosis related studies.Figure 14;Table 11;Reference 77... |
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