Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells

Abstract

Background and Aim. The senescence of human bone marrow mesenchymal stem cells (hBMSCs) can be induced by oxidative stress, but the mechanism by which it occurs is not yet clear. Here, we performed an investigation on the pathogenesis of hypoxia-induced senescence through proteomic analyses and aimed to explore the mechanisms of stem cell senescence. 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.

Identification of Protein Quality Markers in Toad Venom from Bufo gargarizans

“…Liquid chromatographytandem mass spectrometry analysis was performed using a Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). The raw data for each sample were searched using the MASCOT engine (version 2.2; Matrix Science, London, UK) embedded in Proteome Discoverer 1.4 software (Thermo Scientific, Waltham, MA, USA) for identification and quantitation analysis [57][58][59]. The proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with accession number PXD040406.…”

“…To analyze differences in protein expression among groups, a fold change of >1.2 or <0.83 and a p-value of <0.05 were used to identify up and downregulated proteins. NCBI Basic Local Search Tool (BLAST) + client software (NCBI-blast-2.2.28+-win32.exe, Bethesda, Maryland, USA) and InterProScan were used to find homolog sequences for the selected DEPs [58,60]. The GO, Pfam, KEGG, Non-Redundant, and Swiss-Prot databases were used to annotate all sequences using a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 1 November 2022) [56,61].…”

TMT and PRM Based Quantitative Proteomics to Explore the Protective Role and Mechanism of Iristectorin B in Stroke

“…The key differential proteins that were screened for involvement in ferroptosis processes are HMOX1, TFR1, SLC3A2 and NCOA4. These target proteins were further validated via parallel reaction monitoring (PRM) [26][27][28][29][30], the roles of HMOX1, TFR1 and SLC3A in ferroptosis were successfully validated via PRM, and the overall trend of PRM validation results and TMT experimental results were in high agreement, which supported the proteomics data rationality and reliability. Therefore, the identified differentially expressed proteins became the focus of further research into disease resistance mechanisms.…”

Integrated Network Pharmacology and Proteomic Analyses of Targets and Mechanisms of Jianpi Tianjing Decoction in Treating Vascular Dementia

“…Proteomics identified Cox7c to be involved in oxidative phosphorylation and metabolism pathways. Cox7c, a member of the cytochrome c oxidase complex responsible for mitochondrial respiration promotes ATP synthesis and reduces mitochondrial dysfunction [ 59 ]. Recent studies have revealed Cox7c to be a potential biomarker of pathogenesis in Alzheimer's disease [ 60 ].…”