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Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells
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 ana...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416403/ https://www.ncbi.nlm.nih.gov/pubmed/34484348 http://dx.doi.org/10.1155/2021/5555590 |
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author | Mai, Liping He, Guodong Chen, Jing Zhu, Jiening Chen, Shaoxian Hou, Xinghua Yang, Hui Zhang, Mengzhen Wu, Yueheng Lin, Qiuxiong Yang, Min Li, Xiaohong |
author_facet | Mai, Liping He, Guodong Chen, Jing Zhu, Jiening Chen, Shaoxian Hou, Xinghua Yang, Hui Zhang, Mengzhen Wu, Yueheng Lin, Qiuxiong Yang, Min Li, Xiaohong |
author_sort | Mai, Liping |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8416403 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Hindawi |
record_format | MEDLINE/PubMed |
spelling | pubmed-84164032021-09-04 Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells Mai, Liping He, Guodong Chen, Jing Zhu, Jiening Chen, Shaoxian Hou, Xinghua Yang, Hui Zhang, Mengzhen Wu, Yueheng Lin, Qiuxiong Yang, Min Li, Xiaohong Stem Cells Int Research Article 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. Hindawi 2021-08-27 /pmc/articles/PMC8416403/ /pubmed/34484348 http://dx.doi.org/10.1155/2021/5555590 Text en Copyright © 2021 Liping Mai et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Mai, Liping He, Guodong Chen, Jing Zhu, Jiening Chen, Shaoxian Hou, Xinghua Yang, Hui Zhang, Mengzhen Wu, Yueheng Lin, Qiuxiong Yang, Min Li, Xiaohong Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title | Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title_full | Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title_fullStr | Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title_full_unstemmed | Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title_short | Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells |
title_sort | proteomic analysis of hypoxia-induced senescence of human bone marrow mesenchymal stem cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416403/ https://www.ncbi.nlm.nih.gov/pubmed/34484348 http://dx.doi.org/10.1155/2021/5555590 |
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