| Objective: Traditionally,histone acetylation is considered as an important way to regulate epigenetic and regulate gene expression.Recent study found that more than 4000 types of protein could be acetylated,and the modification can affect the stability of the protein,enzyme activities,the interaction between proteins,regulation of biological processes.Previous studies suggest that acetylation enzyme inhibitors can reduce the activation of microglia.The molecular mechanism of effect that inhibiting histone deacetylases(HDAC)by SAHA on microglia will be discussed in this study by the method of systems biology including protein acetylated modification profile,gene expression profile,protein expression profile,and the molecular mechanism of protein acetylated modification will be explored to provides the experimental basis for research and treatment of cerebral ischemia injury and neurodegenerative diseases.Methods: detected the activity of BV2 microglial cells treated by SAHA by MTT.Observe the change of cell morphology after treated by SAHA for different time by microscope.Detected the protein acetylated level of BV2 cells after treating with SAHA by Western blotting.cell proliferation and phagocytosis were detected by Edu test and Annexin V-EGFP BL21.The second generation sequencing was used to analysis the transcriptome of BV2 cells in SAHA affect.Basing on SILAC labeling quantitative proteomic methods was used to analyze acetylated protein profiles and protein expression changes of BV2 cells in SAHA affect.Through the bioinformatics software and methods,such as IPA to analyze the connection between of the three levels of genes,protein and post-translation modification.Results: 1.The MTT test result shows that SAHA inhibited the viability of BV2 in a dose-dependent manner,IC50=9.85.2.Following the increase of SAHA treatment time,the cells had more tentacles gradually,the cellular morphology became more and more similar with activated microglia.3.The result of Western-blotting showed that not only the histone acetylation level was up-regulated after BV2 cells were treated with SAHA for 3 hours to 24 hours,but also the non-histone acetylation level was up-regulated.4.Acetylated proteome analysis showed different expression of acetylated modification in the SAHA-stimulated BV2 cells: 26307 acetylated peptides were detected,28.85% of all peptides,included 3092 acetylated sites.724 acetylated sites were up-regulated and 84 acetylated sites were down-regulated.1215 acetylated proteins were detected,388 proteins only had up-regulated acetylated modification sites,71 proteins only had down-regulated acetylated modification sites and 20 proteins had not only up-regulated acetylated modification sites but also down-regulated acetylated modification sites.The functions of these regulated acetylated protein involved the formation of cytoskeleton,mitochondrial metabolism,gene expression and protein synthesis,degradation.5.RNA-seq result showed that 12123 gene were detected and 444(3.6%)genes were differentially expressing which included 272 genes were up-regulated,172 genes were down-regulated.The up-regulated genes mainly were related to protective immunity,and the down-regulated genes mainly related to combination.6.Proteome analysis showed different expression of proteins in the SAHA-stimulated BV2 cells:5827 proteins were detected,297(5.10%)proteins were differentially expressing which included 151 proteins were up-regulated,146 proteins were down-regulated.The major function of these changed proteins is immune defense.7.The differentially expressed proteins and differentially expressed genes have bigger difference in the functions.And there were only 14 proteins has change in gene expression.There were 10 proteins which both had change on acetylated modification and gene expression,and there were13 proteins which both had change on acetylated modification and protein expression.These results suggested that the short-term effect of SAHA on the cell functions directly regulated the protein acetylated modification level.8.The results of biological function tests:EdU test result showed SAHA could suppress BV2 cells proliferation.Phagocytosis test result showed that the phagocytosis of BV2 cells was strengthened firstly and was weakened later with the increase of SAHA treatment time.These results showed the treatment of single SAHA for short time had an impact on cell biological functions.Conclusion:The histones acetylation enzyme inhibitor SAHA not only regulates the acetylation level of histone,also changes the acetylation level of non-histone.the short-term effect of SAHA on the cell fuctions do not directly regulated gene expression to change the protein expression.The change of protein acetylated modification level provided a molecular basis for cell morphology,biological function,protein expression,the polarization of microglia and gene expression. |
