| ObjectiveVPA(Valproate,VPA)is widely applied for the treatment of epilepsy.In recent years,other pharmacological effects of VPA,such as anti-cancer and the treatment of neurodegeneration diseases,have aroused wide attention of the international medical community.Meanwhile,VPA is a potent teratogen and can induce neural tube defects and cognitive deficits.Therefore,studying the mechanism of VPA contributes greatly in understanding cancer,neuropsychiatric disorders,birth defects,and even effective treatments of those diseases.VPA belongs to the short-chain fatty acid class of histone deacetylase inhibitors and inhibits the activity of class Ⅰ and class Ⅱa histone deacetylases and causes hyperacetylation of histones in vivo and in vitro.Acetylation and deacetylation of histone play an important role in regulation of gene expression,nucleosome assembly and chromatin folding.VPA induces differentiation of various cancer cells and promotes differentiations of neural progenitor cells and osteoblasts.It is suggested that cell fate determination may be related to the inhibition of HD AC.Meanwhile,accurately regulating the differentiation direction of pluripotent stem cells and inccreasing the effectiveness and specificity of differentiation are key issues in clinical application of stem cells.The main purpose of this study is to gain insight into the molecular mechanisms of VPA on differentiation of embryonic carcinoma stem cells,and to analyze related epigenetic and transcription profiles.MethodsIn our study,we selected NCCIT and NT2/D1 embryonic carcinoma cells as our research models.We screened the range of low cytotoxicity of VPA and other HD ACi,including TSA(Trichostatin A,TSA)and CHI(Chidamide,CHI),on NCCIT and NT2/D1 cells by the CCK-8 method.And we induced the differentiation of NCCIT and NT2/D1 cells to somatic cells by 10μM ATRA and subsequently detected the expression levels of stem cell markers Oct4 and Sox2 by Western Blotting and Quantitative Real-time PCR.Later,NT2/D1 and NCCIT cells were treated with VPA,TSA,CHI,and combined with ATRA treatment,respectively.Subsequently we detected the expression levels of Oct4 or Sox2 by Western Blotting and immunofluorescence.NCCIT cells were treated with DMSO or ImM VPA or 10nM TSA for 5 days.Then we analyzed the differences of histone acetylation sites and acetylation levels by liquid chromatography-mass spectrometry(HPLC-MS/MS).At last,the total RNA samples of NT2/D1 cells that treated with DMSO or ImM VPA for 72h were sequenced by RNA-Seq at Illumina Hiseq 4000.Results1.The cytotoxicities of VPA,TSA,and CHI were in a dose-dependent manner in NCCIT and NT2/D1 cells.The low cytotoxic concentration ranges of VPA,TSA and CHI in NCCIT and NT2/D1 were 0.25～2mM,2.5～50nM and 125～1000nM.2.The results of Western Blotting and Immunoflurescence showed that the treatments of VPA,TSA and CHI could promote differentiations of NCCIT and NT2/D1 cells,10μM ATRA combined with VPA or TSA could further promote the disappearance of pluripotency in NT2/D1 cells.3.The quantitative analysis of data of HPLC-MS/MS showed that after the treatment of VPA and TSA,the lysine acetylation levels in H3K23 and H3K27 sites of N-end tail in H3 histone were higher than that of the control group.Besides the lysine acetylation levels in H4K16,other sites of N-end tail in H4 histone H4K8,H4K12,and H4K5 were also selectively higher after VPA treatment but not TSA treatment.4.The transcriptional profiling of NT2/D1 cells reveals that a total of 5018 genes differential expressed genes were affected,including 2723 up-regulated genes and 2295 down-regulated genes after ImM VPA treatment of NT2/D1 cells for 72h.The results of GO and DO enrichment analyses indicated that the biological processes associated with neurodevelopment,angiogenesis,and osteogenesis significantly enriched in NT2/D1 cells.And VPA might cause expression changes in genes related to neural and musculoskeletal diseases.Meanwhile,the KEGG pathway enrichment analysis indicated that the treatment of VPA could affect embryonic carcinoma cells differentiation by stem cell signaling pathways.Besides,VPA might regulated the pluripotency of embryonic carcinoma cells by affecting cell adhesion molecules and extracellular matrix synthesizing,such as glycosaminoglycan and proteoglycans.Conclus ions1.Similar to other HD AC inhibitors,such as TSA and CHI,VPA can promote the differentiations of NT2/D1 and NCCIT pluripotent stem cells into somatic cells.Combining with ATRA treatment,VPA and TSA could further promote the disappearance of pluripotency in NT2/D1 cells.2.Compare with TSA,VPA can selectively increase the levels of acetylation of H4K16,H4K8,H4K12 and H4K5 sites in the H4 N-Terminal tail of NCCIT cells.It provides an epigenetic basis for further understanding of the molecular mechanism of VPA that involved in cell fate determination.3.The transcriptional profiling of NT2/D1 cells reveals that VPA can inhibit expression of the classic maintenance of pluripotent genes,while activating the development of neural,hematopoietic system,and cell adhesion related genes.Moreover,VPA could affect pluripotent stem cells differentiation by inducing stem cell signaling pathway.Besides,VPA might regulated pluripotency of embryonic carcinoma cells by affecting cell adhesion molecules and extracellular matrix synthesizing,such as glycosaminoglycan and proteoglycans. |
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