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The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP-6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t-test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)-382 and miR-203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet-derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR-495, miR-376a and miR-543), the upregulated miR-106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N-acetylgalactosaminyltransferase 1 and acyl-CoA synthetase long-chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR-203 and miR-382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR-495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR-376a, miR-543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.

Background: The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs. Methods: We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3′-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation. Results: We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3′-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone. Conclusions: Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

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