HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC ...HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma Ⅰ, Sal Ⅰ, Xho Ⅰ and Hind III which are sensitive to 5-methylcytosirte were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.展开更多
The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely enc...The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely encoded in the epigenome.Recent advances in single-cell multimodality epigenomic technologies facilitate directly interrogating the reg-ulatory relationship between multi-layer molecular information in the same cell.In this review,we discuss recent progress in development of single-cell multimodality epigenomic technologies and applications in elucidating cellular diversifications in development and diseases,with a focus on protein-DNA interactomics and regulatory links between epigenome and tran-scriptome.Further,we provide perspective on the future direction of single-cell multiomics tool development as well as challenges facing ahead.展开更多
目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构...目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。展开更多
目的探讨微小RNA-214-5p(miR-214-5p)对神经母细胞瘤(NB)DNA损伤和化疗敏感性的调控作用及机制。方法通过TARGET数据库分析范可尼贫血互补组A型(fanconi anemia complementary group A protein,FACNA)蛋白与NB临床病理特征的关系。构建m...目的探讨微小RNA-214-5p(miR-214-5p)对神经母细胞瘤(NB)DNA损伤和化疗敏感性的调控作用及机制。方法通过TARGET数据库分析范可尼贫血互补组A型(fanconi anemia complementary group A protein,FACNA)蛋白与NB临床病理特征的关系。构建miR-214-5p低表达、FANCA过/低表达NB细胞系,采用细胞计数试剂盒-8(CCK-8)、移植瘤模型检测其表达对NB体内外增殖的影响,并验证miR-214-5p与FANCA靶向关系。结果GEO数据库和TARGET数据库中筛查出与NB相关的交集基因为FANCA,其表达与调节细胞增殖基因、预后及临床病理特征密切相关(P<0.05)。与空载体(Vector)组比较,FANCA组细胞活力、移植瘤体积和重量升高(P<0.05);与shNC组比较,shFANCA1组、shFANCA2组细胞活力降低(P<0.05);与对照组比较,1 mmol/L过氧化氢(H_(2)O_(2))处理后磷酸化组蛋白γ-H2AX升高(P<0.05),但FANCA过表达后γ-H2AX降低(P<0.05)。随着多柔比星浓度增加,细胞活力降低,但FANCA组细胞活力高于Vector组(P<0.05)。miR-214-5p与FANCA存在靶向关系。结论miR-214-5p可靶向降低FANCA表达抑制NB细胞增殖,参与细胞DNA损伤和化疗敏感性调控。展开更多
文摘HL-60 cells have been induced with differentiation index 16% by S-adenosyl-L-methionine (SAM) as in-ducer in the presence of optimum concentration of 10 μmol/L. The methylation level of genome DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma Ⅰ, Sal Ⅰ, Xho Ⅰ and Hind III which are sensitive to 5-methylcytosirte were used to cleave the genome DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.
基金supported by the National Key Research and Development Program of China(2021YFA1100101 and 2017YFA0103402)the National Natural Science Foundation of China(32025015 and 31771607)the Peking-Tsinghua Center for Life Sciences.
文摘The emergence of single-cell genomic and transcriptomic sequencing accelerates the development of single-cell epigenomic technologies,providing an unprecedented opportunity for decoding cell fate decisions largely encoded in the epigenome.Recent advances in single-cell multimodality epigenomic technologies facilitate directly interrogating the reg-ulatory relationship between multi-layer molecular information in the same cell.In this review,we discuss recent progress in development of single-cell multimodality epigenomic technologies and applications in elucidating cellular diversifications in development and diseases,with a focus on protein-DNA interactomics and regulatory links between epigenome and tran-scriptome.Further,we provide perspective on the future direction of single-cell multiomics tool development as well as challenges facing ahead.
文摘目的探讨乙型肝炎病毒DNA聚合酶反式调节蛋白1(HBV DNA polymerase trans activated protein 1,HBVDNAPTP1)在羧酸酯酶1(carboxylesterase 1,CES1)介导的单核细胞凋亡信号通路中的作用机制。方法利用Phyre2在线工具预测得到CES1三级结构,分别用pCMV-Myc载体构建HBVDNAPTP1基因、pCMV-HA载体构建CES1基因及其截短体219-264aa和265-303aa的真核细胞表达质粒。分别转染细胞后通过免疫荧光和免疫共沉淀法确定HBVDNAPTP1和CES1互相作用的区段。后通过HBVDNAPTP1和CES1单独转染及共转染,检测其对细胞凋亡率的影响;同时在分离人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)上转染上述质粒,以确定CES1和HBVDNAPTP1对单核细胞形态的影响。利用qRT-PCR和Western blot检测不同质粒转染后下游Janus激酶-1(Janus kinase,JAK1)和信号传导及转录激活蛋白3(Signal transducer and activator of transcription 3,STAT3)的基因和蛋白的表达情况。结果HBVDNAPTP1与CES1有靶向关系,其中HBVDNAPTP1与CES1全长靶向结合效果最好,故后续实验以CES1全长序列为对象开展。CCK-8和PBMC电镜实验结果表明,与Control组和NC组相比,ov-HBVDNAPTP1组、ov-CES1组和ov-HBVDNAPTP1+ov-CES1组THP-1细胞的凋亡率均升高(P<0.001),细胞受损;与ov-HBVDNAPTP1组和ov-CES1组相比,ov-HBVDNAPTP1+ov-CES1组细胞凋亡率进一步升高(P<0.01),凋亡细胞、坏死细胞的数量增多。qRT-PCR和Western blot结果显示,HBVDNAPTP1和CES1均可以在转录水平和翻译水平上显著上调JAK1和STAT3的表达,且二者同时作用时上调趋势更加明显。结论本研究证实HBVDNAPTP1与CES1二者可协同抑制THP-1细胞增殖并诱导细胞损伤;还可协同上调JAK1/STAT3信号通路在转录和蛋白水平的表达来调控单核细胞凋亡。该发现为深入理解HBV感染相关免疫调控机制提供了新的理论依据。
文摘目的探讨微小RNA-214-5p(miR-214-5p)对神经母细胞瘤(NB)DNA损伤和化疗敏感性的调控作用及机制。方法通过TARGET数据库分析范可尼贫血互补组A型(fanconi anemia complementary group A protein,FACNA)蛋白与NB临床病理特征的关系。构建miR-214-5p低表达、FANCA过/低表达NB细胞系,采用细胞计数试剂盒-8(CCK-8)、移植瘤模型检测其表达对NB体内外增殖的影响,并验证miR-214-5p与FANCA靶向关系。结果GEO数据库和TARGET数据库中筛查出与NB相关的交集基因为FANCA,其表达与调节细胞增殖基因、预后及临床病理特征密切相关(P<0.05)。与空载体(Vector)组比较,FANCA组细胞活力、移植瘤体积和重量升高(P<0.05);与shNC组比较,shFANCA1组、shFANCA2组细胞活力降低(P<0.05);与对照组比较,1 mmol/L过氧化氢(H_(2)O_(2))处理后磷酸化组蛋白γ-H2AX升高(P<0.05),但FANCA过表达后γ-H2AX降低(P<0.05)。随着多柔比星浓度增加,细胞活力降低,但FANCA组细胞活力高于Vector组(P<0.05)。miR-214-5p与FANCA存在靶向关系。结论miR-214-5p可靶向降低FANCA表达抑制NB细胞增殖,参与细胞DNA损伤和化疗敏感性调控。
