Isocitrate dehydrogenase 2(IDH2)and glutamate dehydrogenase 1(GLUD1)are key enzymes involved in the production ofα-ketoglutarate(α-KG),a metabolite central to the tricarboxylic acid cycle and glutamine metabolism.In...Isocitrate dehydrogenase 2(IDH2)and glutamate dehydrogenase 1(GLUD1)are key enzymes involved in the production ofα-ketoglutarate(α-KG),a metabolite central to the tricarboxylic acid cycle and glutamine metabolism.In this study,we investigated the impact of IDH2 and GLUD1 on early porcine embryonic development following IDH2 and GLUD1 knockdown(KD)via doublestranded RNA(dsRNA)microinjection.Results showed that KD reducedα-KG levels,leading to delayed embryonic development,decreased blastocyst formation,increased apoptosis,reduced blastomere proliferation,and pluripotency.Additionally,IDH2 and GLUD1 KD induced abnormally high levels of trimethylation of lysine 20 of histone H4(H4K20me3)at the 4-cell stage,likely resulting in transcriptional repression of embryonic genome activation(EGA)-related genes.Notably,KD of lysine methyltransferase 5C(KMT5C)and supplementation with exogenousα-KG reduced H4K20me3 expression and partially rescued these defects,suggesting a critical role of IDH2 and GLUD1 in the epigenetic regulation and proper development of porcine embryos.Overall,this study highlights the significance of IDH2 and GLUD1 in maintaining normal embryonic development through their influence onα-KG production and subsequent epigenetic modifications.展开更多
Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analy...Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines.Pyrophosphate sequencing was performed to determine the methylation degree.Then,the enrichment of H4K20mel and H3K9ac was determined using ChIP-qPCR.Flow cytometry was used to analyze the cell cycle.Results:The IC_(50) of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line.Genistein upregulated H4K20mel,KMT5A and Wnt suppressor genes,including Wnt5a,and downregulated the downstream target genes of Wnt,such as c-myc and β-catenin.The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged.However,the enrichment of H4K20mel in the Wnt5a promoter and coding regions increased.In addition,genistein upregulated Phospho-cdc2,Mytl,Cyclin A,Cyclin E2,p21 and Phospho-histone H3,but downregulated Phospho-weel.Cell cycle arrest was induced in the G2/M phase.Conclusion:Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20mel in the Wnt5a gene promoter and coding regions,rather than demethylation.Genistein also blocks the cell cycle in the G2/M phase.Therefore,genistein is a potential anti-leukemia drug.展开更多
目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 ...目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 h。采用MTT法测定Ha Ca T细胞的存活情况。另设对照(24 h)组、亚砷酸钠(10μmol/L,24 h)染毒组、PR-Set7小干扰RNA干扰组(Si PR-Set7,50 nmol/L,48 h)和联合暴露(50 nmol/L Si PR-Set7干扰48 h后,10μmol/L亚砷酸钠暴露24 h)组。分别采用q RT-PCR法检测PR-Set7 m RNA的表达水平,采用Western blot法检测H4K20me1和PR-Set7蛋白的表达水平,采用单细胞凝胶电泳法检测DNA损伤水平。结果与对照组相比,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞的存活率均较低,差异有统计学意义(P<0.05)。与对照组比较,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均升高,而H4K20me1蛋白的表达水平降低;2.5、5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞尾部DNA%及尾矩升高,差异有统计学意义(P<0.05)。随着亚砷酸钠染毒浓度的升高,Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均呈上升趋势,而H4K20me1蛋白和m RNA的表达水平呈下降趋势,DNA损伤水平(尾部DNA%及尾矩)呈上升趋势。与对照组比较,Si PR-Set7干扰组Ha Ca T细胞PR-Set7蛋白的表达大幅下降;亚砷酸钠染毒组、Si PRSet7干扰组和联合暴露组Ha Ca T细胞H4K20me1蛋白的表达水平均降低,而尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。与亚砷酸钠染毒组比较,联合暴露组Ha Ca T细胞PR-Set7蛋白和m RNA以及H4K20me1蛋白的表达均降低,而Ha Ca T细胞尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。结论在对Ha Ca T细胞进行PR-Set7干扰后,亚砷酸钠可能通过抑制组蛋白甲基转移酶PR-Set7蛋白及m RNA的表达水平,从而降低H4K20me1的修饰水平,影响Ha Ca T细胞DNA损伤修复能力,使DNA损伤加重。展开更多
基金supported by the National Research Foundation(NRF)of Korea grant funded by the Korean government(MSIT)(2022R1A2C300769),Republic of Korea。
文摘Isocitrate dehydrogenase 2(IDH2)and glutamate dehydrogenase 1(GLUD1)are key enzymes involved in the production ofα-ketoglutarate(α-KG),a metabolite central to the tricarboxylic acid cycle and glutamine metabolism.In this study,we investigated the impact of IDH2 and GLUD1 on early porcine embryonic development following IDH2 and GLUD1 knockdown(KD)via doublestranded RNA(dsRNA)microinjection.Results showed that KD reducedα-KG levels,leading to delayed embryonic development,decreased blastocyst formation,increased apoptosis,reduced blastomere proliferation,and pluripotency.Additionally,IDH2 and GLUD1 KD induced abnormally high levels of trimethylation of lysine 20 of histone H4(H4K20me3)at the 4-cell stage,likely resulting in transcriptional repression of embryonic genome activation(EGA)-related genes.Notably,KD of lysine methyltransferase 5C(KMT5C)and supplementation with exogenousα-KG reduced H4K20me3 expression and partially rescued these defects,suggesting a critical role of IDH2 and GLUD1 in the epigenetic regulation and proper development of porcine embryos.Overall,this study highlights the significance of IDH2 and GLUD1 in maintaining normal embryonic development through their influence onα-KG production and subsequent epigenetic modifications.
基金supported by grants from the National Natural Science Foundation of China(No.81800167)the Natural Science Foundation of Fujian Province(No.2017J05132)+4 种基金the Fujian Provincial Health Technology Project(No.2018-ZQN-40)the Start-up Fund Project of Fujian Medical University(No.2016QH020)the Construction Project of Fujian Medical Center of Hematology(No.Min201704)the National and Fujian Provincial Key Clinical Specialty Discipline Construction Program,ChinaClinical Research Center for Hematological Malignancies of Fujian Province.
文摘Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines.Pyrophosphate sequencing was performed to determine the methylation degree.Then,the enrichment of H4K20mel and H3K9ac was determined using ChIP-qPCR.Flow cytometry was used to analyze the cell cycle.Results:The IC_(50) of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line.Genistein upregulated H4K20mel,KMT5A and Wnt suppressor genes,including Wnt5a,and downregulated the downstream target genes of Wnt,such as c-myc and β-catenin.The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged.However,the enrichment of H4K20mel in the Wnt5a promoter and coding regions increased.In addition,genistein upregulated Phospho-cdc2,Mytl,Cyclin A,Cyclin E2,p21 and Phospho-histone H3,but downregulated Phospho-weel.Cell cycle arrest was induced in the G2/M phase.Conclusion:Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20mel in the Wnt5a gene promoter and coding regions,rather than demethylation.Genistein also blocks the cell cycle in the G2/M phase.Therefore,genistein is a potential anti-leukemia drug.
文摘目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 h。采用MTT法测定Ha Ca T细胞的存活情况。另设对照(24 h)组、亚砷酸钠(10μmol/L,24 h)染毒组、PR-Set7小干扰RNA干扰组(Si PR-Set7,50 nmol/L,48 h)和联合暴露(50 nmol/L Si PR-Set7干扰48 h后,10μmol/L亚砷酸钠暴露24 h)组。分别采用q RT-PCR法检测PR-Set7 m RNA的表达水平,采用Western blot法检测H4K20me1和PR-Set7蛋白的表达水平,采用单细胞凝胶电泳法检测DNA损伤水平。结果与对照组相比,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞的存活率均较低,差异有统计学意义(P<0.05)。与对照组比较,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均升高,而H4K20me1蛋白的表达水平降低;2.5、5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞尾部DNA%及尾矩升高,差异有统计学意义(P<0.05)。随着亚砷酸钠染毒浓度的升高,Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均呈上升趋势,而H4K20me1蛋白和m RNA的表达水平呈下降趋势,DNA损伤水平(尾部DNA%及尾矩)呈上升趋势。与对照组比较,Si PR-Set7干扰组Ha Ca T细胞PR-Set7蛋白的表达大幅下降;亚砷酸钠染毒组、Si PRSet7干扰组和联合暴露组Ha Ca T细胞H4K20me1蛋白的表达水平均降低,而尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。与亚砷酸钠染毒组比较,联合暴露组Ha Ca T细胞PR-Set7蛋白和m RNA以及H4K20me1蛋白的表达均降低,而Ha Ca T细胞尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。结论在对Ha Ca T细胞进行PR-Set7干扰后,亚砷酸钠可能通过抑制组蛋白甲基转移酶PR-Set7蛋白及m RNA的表达水平,从而降低H4K20me1的修饰水平,影响Ha Ca T细胞DNA损伤修复能力,使DNA损伤加重。
