Mai Liping, He Guodong, Chen Jing, Zhu Jiening, Chen Shaoxian, Hou Xinghua, Yang Hui, Zhang Mengzhen, Wu Yueheng, Lin Qiuxiong, Yang Min, Li Xiaohong
Research Department of Medical Sciences, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Guangdong Provincial Key Laboratory of South China Structural Heart Disease, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Stem Cells Int. 2021 Aug 27;2021:5555590. doi: 10.1155/2021/5555590. eCollection 2021.
Hypoxia in hBMSCs was induced for 0, 4, and 12 hours, and cellular senescence was evaluated by senescence-associated -galactosidase (SA--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).
Cell senescence detected by SA--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.
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.
将人骨髓间充质干细胞（hBMSCs）诱导缺氧0、4和12小时，通过衰老相关β-半乳糖苷酶（SA-β-gal）染色评估细胞衰老。采用串联质谱标签（TMT）标记结合液相色谱-串联质谱（LC-MS/MS）对hBMSCs缺氧进行差异蛋白质组学分析。运用平行反应监测（PRM）分析验证候选蛋白。通过蛋白质印迹法评估信号通路的验证情况。采用膜联蛋白V/7-氨基放线菌素D（7-AAD）染色，通过流式细胞术评估细胞凋亡。使用荧光探针2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）检测活性氧（ROS）的产生。
SA-β-gal活性检测显示，12小时缺氧诱导组的细胞衰老程度更高。对12小时缺氧诱导细胞进行TMT分析，鉴定出6000多种蛋白质，其中包括686种差异表达蛋白质。基于生物学通路分析，我们发现衰老相关蛋白主要富集于癌症通路、PI3K-Akt通路和细胞衰老信号通路。细胞周期蛋白依赖性激酶1（CDK1）、细胞周期蛋白依赖性激酶2（CDK2）和细胞周期蛋白D1（CCND1）是蛋白质-蛋白质相互作用（PPI）分析中的重要节点。此外，通过PRM验证了CCND1、泛醌还原酶同源物（UQCRH）和细胞色素c氧化酶亚基ⅦC（COX7C）的表达。hBMSCs缺氧诱导12小时降低了CCND1表达，但促进了ROS产生和细胞凋亡。PI3K激动剂740 Y-P可显著减轻这些效应，而LY294002可使其减弱。
hBMSCs缺氧通过激活PI3K依赖性信号通路抑制CCND1表达，但促进ROS产生和细胞凋亡。这些发现详细描述了与hBMSCs缺氧诱导衰老相关的蛋白质组学特征，有助于我们理解导致干细胞衰老的分子机制。
