Quantitative proteomic analysis of epidermal stem cells in oxygen-glucose deprivation conditions
- ZHANG Huayu, YIN Siyuan, LIU Jian, MA Jiaxu, SONG Ru, CAO Guoqi, WANG Yibing
Journal of Shandong University (Health Sciences). 2021, 59(4):
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Objective To find the key proteins and pathways leading to the dysfunction of epidermal stem cells in oxygen-glucose deprivation(OGD)conditions and to explore the formation mechanism of chronic hard healing wounds. Methods Epidermal stem cells were extracted from newborn mice and cultured normally to the P3 generation. The cells were cultured under OGD conditions for 0, 3, 6, 9 and 12 h, respectively, and then were divided into five goups: 0 h was taken as the control group, and the other groups were taken as the experimental groups. There are four comparison groups: OGD3_vs_OGD0, OGD6_vs_OGD0, OGD9_vs_OGD0 and OGD12_vs_OGD0. Quantitative proteomics determination was performed on different groups of epidermal stem cells using Tandem mass tag(TMT)technology. The four comparison groups were screened for proteins with significantly differential expression. These differentially expressed proteins(DEPs)were analyzed for Gene Ontology(GO)category, KEGG pathway, protein-protein interaction(PPI), gene overlap, and analysis of pathways and proteins related to mitophagy to find the key proteins and pathways in the dysfunction of epidermal stem cells. Results A total of 4 852 quantifiable proteins were detected in the experiment. In the four comparison groups, that is, OGD3_vs_OGD0, OGD6_vs_OGD0, OGD9_vs_OGD0, and OGD12_vs_OGD0, the number of up-regulated DEPs were 1, 225, 346 and 386, respectively, while the number of down-regulated DEPs were 26, 229, 330 and 462, respectively. In the analysis of the comparison group OGD6_vs_OGD0, key pathways included “ribonucleoprotein complex biogenesis(GO: 0022613)”“chromatin binding(GO: 0003682)”“cytoplasmic ribosomal large subunit(GO:0022625)” and “ribosome(ko03010)”,and the 21 hub proteins screened through PPI analysis were all structural proteins of the ribosome, of which the most important seed protein was the mitochondrial ribosomal protein MRPL24. In the comprehensive analysis of the four comparison groups, it was found that there was a large amount of overlap between the DEPs of each comparison group, and key pathways were all related to protein processing. In the analysis of mitophagy-related pathway and proteins, it was found that the expression of Tbc1d15, Rab7a, which were related to lysosomal maturation and p53, continued to be up-regulated, while the expression of LC3 related to the level of autophagy flow did not show a definite trend. Conclusion The key protein “MRPL24” obtained through TMT quantitative proteomics analysis and the pathways related to protein processing are the key to the dysfunction of epidermal stem cells in OGD conditions. They will be the breakthrough points for further research on the formation mechanism of chronic hard healing wounds.