Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expre...Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expression from Dnmt3b to Dnmt3a during mouse embryogenesis and in tissue-specific stem cells, suggesting distinct functions of Dnmt3a and Dnmt3b during these processes. In this study, to characterize the functions of Dnmt3a and Dnmt3b in pluripotent stem cells, we exogenously transfected ES cells with Dnmt3a and Dnmt3b cDNAs linked to an internal ribosome entry site-green fluorescent protein gene, and then analyzed the effects of expression of these de novo DNA methyltransferases on ES cell growth and differentiation. ES cells expressing Dnmt3b showed specific downregulation of pluripotency marker genes such as Nanog and Oct 3/4. In addition, Dnmt3a-transfected ES cells showed a specific increase in mitotic index, while Dnmt3b-transfected ES cells showed a decrease in mitotic index. These results suggest that Dnmt3b has important physiological roles in the initial process of stem cell differentiation and that Dnmt3a has a function in stem cell proliferation.展开更多
目的 探讨乳腺癌患者血清核受体亚家族C组成员2(nuclear receptor subfamily 3, group C, member 2,NR3C2),DNA甲基化转移酶-3A[DNA(cytosine-5)-methyltransferase 3A,DNMT3A]水平及其临床诊断价值。方法收集复旦大学附属华东医院2017...目的 探讨乳腺癌患者血清核受体亚家族C组成员2(nuclear receptor subfamily 3, group C, member 2,NR3C2),DNA甲基化转移酶-3A[DNA(cytosine-5)-methyltransferase 3A,DNMT3A]水平及其临床诊断价值。方法收集复旦大学附属华东医院2017年5月~2020年12月期间住院的94例乳腺癌患者为乳腺癌组,另选取同期健康体检者86例作为对照组。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测两组血清NR3C2和DNMT3A水平,Pearson法分析乳腺癌患者血清NR3C2和DNMT3A表达水平相关性,多因素Logistic回归分析乳腺癌发生的影响因素,受试者工作特征(receiver operating characteristic,ROC)曲线评估NR3C2和DNMT3A对乳腺癌的诊断价值。结果 乳腺癌组患者血清NR3C2水平为317.84±33.47 ng/L,低于对照组(374.25±47.72ng/L),DNMT3A的表达水平为451.63±75.47μg/L,高于对照组(349.85±63.72μg/L),差异有统计学意义(t=9.243,9.729,均P<0.05)。Pearson分析结果显示,乳腺癌患者血清NR3C2与DNMT3A表达水平存在明显负相关(r=-0.501,P=0.000)。Logistic回归分析结果显示,NR3C2高水平为乳腺癌发生的保护因素(OR=0.563,95%CI:0.372~0.851,P=0.006),DNMT3A高水平是乳腺癌发生的危险因素(OR=1.834,95%CI:1.249~2.693,P=0.002)。ROC曲线结果发现,血清NR3C2与DNMT3A诊断乳腺癌的曲线下面积(area under the curve,AUC)分别为0.853(0.791~0.915),0.930(0.896~0.965),当两者联合时其AUC为0.969(0.949~0.990),高于两者单独检测(Z=3.460,1.894,P=0.000,0.034)。结论 乳腺癌患者血清NR3C2表达水平降低,DNMT3A表达水平升高,两者联合检测能够提高乳腺癌的诊断价值。展开更多
Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effec...Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.展开更多
急性髓系白血病（AML）是一组异质性恶性血液系统疾病，无论在形态学、免疫学、细胞遗传学、分子生物学及临床特点上都存在很大的差异。随着基因测序技术逐步成熟与推广，目前发现与AML相关的分子异常除Ⅰ类突变（主要是信号传导分子）...急性髓系白血病（AML）是一组异质性恶性血液系统疾病，无论在形态学、免疫学、细胞遗传学、分子生物学及临床特点上都存在很大的差异。随着基因测序技术逐步成熟与推广，目前发现与AML相关的分子异常除Ⅰ类突变（主要是信号传导分子）和Ⅱ类突变（主要是转录因子）外，表观遗传调节对造血干细胞分化过程及功能也起着举足轻重的作用，与AML中表观遗传学有关的基因突变包括异柠檬酸脱氢酶-1（IDH1）、异柠檬酸脱氢酶-2（IDH2）、TET 家族羟化酶2（TET2）、混合系白血病基因（MLL）和蛋白转导结构域（PTD）突变等［1］。而甲基化转移酶3A（DNMT3A）基因突变是近年来在AML患者中发现的与表观遗传调节有关的突变，该基因的突变影响DNA甲基化水平，因此该基因突变可能在 AML 患者发病过程中起着非常重要的作用。现简要总结近年来与DNM T3A突变有关的研究结果。展开更多
目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得...目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA 3.1(+)的多克隆位点,构建反义DNM T 3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNM T 3b基因片断真核表达载体可为进一步研究DNM T 3b基因功能提供实验工具。展开更多
文摘Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, have been identified in humans and mice to contribute to the methylation of unmodified DNA. We recently showed a transition of de novo DNA methyltransferase expression from Dnmt3b to Dnmt3a during mouse embryogenesis and in tissue-specific stem cells, suggesting distinct functions of Dnmt3a and Dnmt3b during these processes. In this study, to characterize the functions of Dnmt3a and Dnmt3b in pluripotent stem cells, we exogenously transfected ES cells with Dnmt3a and Dnmt3b cDNAs linked to an internal ribosome entry site-green fluorescent protein gene, and then analyzed the effects of expression of these de novo DNA methyltransferases on ES cell growth and differentiation. ES cells expressing Dnmt3b showed specific downregulation of pluripotency marker genes such as Nanog and Oct 3/4. In addition, Dnmt3a-transfected ES cells showed a specific increase in mitotic index, while Dnmt3b-transfected ES cells showed a decrease in mitotic index. These results suggest that Dnmt3b has important physiological roles in the initial process of stem cell differentiation and that Dnmt3a has a function in stem cell proliferation.
文摘目的 探讨乳腺癌患者血清核受体亚家族C组成员2(nuclear receptor subfamily 3, group C, member 2,NR3C2),DNA甲基化转移酶-3A[DNA(cytosine-5)-methyltransferase 3A,DNMT3A]水平及其临床诊断价值。方法收集复旦大学附属华东医院2017年5月~2020年12月期间住院的94例乳腺癌患者为乳腺癌组,另选取同期健康体检者86例作为对照组。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测两组血清NR3C2和DNMT3A水平,Pearson法分析乳腺癌患者血清NR3C2和DNMT3A表达水平相关性,多因素Logistic回归分析乳腺癌发生的影响因素,受试者工作特征(receiver operating characteristic,ROC)曲线评估NR3C2和DNMT3A对乳腺癌的诊断价值。结果 乳腺癌组患者血清NR3C2水平为317.84±33.47 ng/L,低于对照组(374.25±47.72ng/L),DNMT3A的表达水平为451.63±75.47μg/L,高于对照组(349.85±63.72μg/L),差异有统计学意义(t=9.243,9.729,均P<0.05)。Pearson分析结果显示,乳腺癌患者血清NR3C2与DNMT3A表达水平存在明显负相关(r=-0.501,P=0.000)。Logistic回归分析结果显示,NR3C2高水平为乳腺癌发生的保护因素(OR=0.563,95%CI:0.372~0.851,P=0.006),DNMT3A高水平是乳腺癌发生的危险因素(OR=1.834,95%CI:1.249~2.693,P=0.002)。ROC曲线结果发现,血清NR3C2与DNMT3A诊断乳腺癌的曲线下面积(area under the curve,AUC)分别为0.853(0.791~0.915),0.930(0.896~0.965),当两者联合时其AUC为0.969(0.949~0.990),高于两者单独检测(Z=3.460,1.894,P=0.000,0.034)。结论 乳腺癌患者血清NR3C2表达水平降低,DNMT3A表达水平升高,两者联合检测能够提高乳腺癌的诊断价值。
基金supported by the National Natural Science Foundation of China,No.82171270 (to ZL)Public Service Platform for Artificial In telligence Screening and Auxiliary Diagnosis for the Medical and Health Industry,Ministry of Industry and Information Technology of the People's Republic of China,No.2020-0103-3-1 (to ZL)+3 种基金the Natural Science Foundation of Beijing,No.Z200016 (to ZL)Beijing Talents Project,No.2018000021223ZK03 (to ZL)Beijing Municipal Committee of Science and Technology,No.Z201 100005620010 (to ZL)CAMS Innovation Fund for Medical Sciences,No.2019-I2M-5-029 (to YongW)。
文摘Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.
文摘急性髓系白血病（AML）是一组异质性恶性血液系统疾病，无论在形态学、免疫学、细胞遗传学、分子生物学及临床特点上都存在很大的差异。随着基因测序技术逐步成熟与推广，目前发现与AML相关的分子异常除Ⅰ类突变（主要是信号传导分子）和Ⅱ类突变（主要是转录因子）外，表观遗传调节对造血干细胞分化过程及功能也起着举足轻重的作用，与AML中表观遗传学有关的基因突变包括异柠檬酸脱氢酶-1（IDH1）、异柠檬酸脱氢酶-2（IDH2）、TET 家族羟化酶2（TET2）、混合系白血病基因（MLL）和蛋白转导结构域（PTD）突变等［1］。而甲基化转移酶3A（DNMT3A）基因突变是近年来在AML患者中发现的与表观遗传调节有关的突变，该基因的突变影响DNA甲基化水平，因此该基因突变可能在 AML 患者发病过程中起着非常重要的作用。现简要总结近年来与DNM T3A突变有关的研究结果。
文摘目的构建反义DNA甲基化酶3b(DNM T 3b)基因片断真核表达载体,为研究DNM T 3b基因功能提供工具。方法根据DNM T 3b基因cDNA序列中编码序列设计PCR引物,在上下游引物5′端分别添加X baⅠ和K pnⅠ酶切位点,RT-PCR从胆管癌细胞QBC-939中获得485bp的DNA片断;将该片断反向插入真核表达载体pcDNA 3.1(+)的多克隆位点,构建反义DNM T 3b基因片断真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到467bp特异条带,双酶切鉴定得到471bp片断和5.4kb载体片断,DNA测序说明插入片断序列正确。结论本研究构建的反义DNM T 3b基因片断真核表达载体可为进一步研究DNM T 3b基因功能提供实验工具。
