Identification of differentially expressed autophagy genes associated with osteogenic differentiation in human bone marrow mesenchymal stem cells

Background: Mesenchymal stem cells derived from human tissues have been widely used for tissue re-generation because of their strong self-renewal capacity and multi-potential properties. Autophagy plays a vital role in maintaining bone homeostasis. However, the mechanism underlying this role for autophagy in the osteogenic differentiation of mesenchymal stem cells remains to be elucidated. Methods: Two microarray datasets were down-loaded from the GEO database. Fourteen bone marrow mesenchymal stem cell samples comprising control and induction groups were selected to identify differentially expressed autophagy-related genes via multiple bioinformatics approaches, followed by functional analysis. Interactions among differentially expressed autophagy genes, miRNAs, and transcription factors were analyzed and visualized using Cytoscape software. The association between hub differentially expressed genes and autophagy was validated by qRT-PCR. Results: Ten autophagy-related genes (including VPS8, NDRG4, and CYBB) were identified as osteogenic hub genes. Correlation analysis revealed that CYBB was highly correlated with the sensitivity to multiple drugs, such as imexon, megestrol acetate, and isotretinoin. The regulatory network displayed a complex connection among miRNAs, transcription factors, and differentially expressed autophagy genes. Friends' analysis showed that NDRG4 was highly closely related to other hub genes (P < 0.05). Furthermore, NDRG4 expression was downregulated in the induction group (P < 0.01). NDRG4 was significantly correlated with infiltrating immune cells, including monocytes, eosinophils, type 17 T helper cells, neutrophils, activated CD8 T cells, and immature B cells. Levels of the 10 autophagy-related genes (including VPS8, NDRG4, and CYBB) were successfully validated based on in vitro experiments. Conclusion: We identified candidate molecules to further investigate their functions in osteogenesis, providing novel insights into the role of autophagy in mesenchymal stem cell differentiation.