Abstract
Methods Hypoxia in hBMSCs was induced for 0, 4, and 12 hours, and cellular senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining. Tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of hypoxia in hBMSCs. Parallel reaction monitoring (PRM) analysis was used to validate the candidate proteins. Verifications of signaling pathways were evaluated by western blotting. Cell apoptosis was evaluated using Annexin V/7-AAD staining by flow cytometry. The production of reactive oxygen species (ROS) was detected by the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Results Cell senescence detected by SA-β-gal activity was higher in the 12-hour hypoxia-induced group. TMT analysis of 12-hour hypoxia-induced cells identified over 6000 proteins, including 686 differentially expressed proteins. Based on biological pathway analysis, we found that the senescence-associated proteins were predominantly enriched in the cancer pathways, PI3K-Akt pathway, and cellular senescence signaling pathways. CDK1, CDK2, and CCND1 were important nodes in PPI analyses. Moreover, the CCND1, UQCRH, and COX7C expressions were verified by PRM. Hypoxia induction for 12 hours in hBMSCs reduced CCND1 expression but promoted ROS production and cell apoptosis. Such effects were markedly reduced by the PI3K agonist, 740 Y-P, and attenuated by LY294002. Conclusions Hypoxia of hBMSCs inhibited CCND1 expression but promoted ROS production and cell apoptosis through activating the PI3K-dependent signaling pathway. These findings provided a detailed characterization of the proteomic profiles related to hypoxia-induced senescence of hBMSCs and facilitated our understanding of the molecular mechanisms leading to stem cell senescence.
Highlights
Mesenchymal stem cells (MSCs) have become an important resource for cell therapy and regenerative medicine [1] due to their self-renewal capacity, multidirectional differentiation, and low immunogenicity
Proteomic analyses based on Tandem mass tag (TMT) identified 686 proteins, of which 400 proteins were upregulated and 286 were downregulated in the hypoxia-induced group (12 h) (Figure 3). 302 significantly differentially expressed proteins were identified, of which 227 were upregulated and 75 were downregulated after hypoxia induction for 4 hours (Figure S2)
The collective results suggested that dramatic changes in gene expression occur during human bone marrow mesenchymal stem cells (hBMSCs) senescence
Summary
Mesenchymal stem cells (MSCs) have become an important resource for cell therapy and regenerative medicine [1] due to their self-renewal capacity, multidirectional differentiation, and low immunogenicity. Promoting MSC proliferation and chondrogenic differentiation in cartilage can repair osteoarthritic lesions via activating the PI3K-Akt signaling pathway [7]. Hypoxia induction for 12 hours in hBMSCs reduced CCND1 expression but promoted ROS production and cell apoptosis. Such effects were markedly reduced by the PI3K agonist, 740 Y-P, and attenuated by LY294002. Hypoxia of hBMSCs inhibited CCND1 expression but promoted ROS production and cell apoptosis through activating the PI3K-dependent signaling pathway. These findings provided a detailed characterization of the proteomic profiles related to hypoxia-induced senescence of hBMSCs and facilitated our understanding of the molecular mechanisms leading to stem cell senescence
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