反义DNMT1、DNMT3b基因真核表达载体联合转染对人胆管癌细胞增殖和凋亡的影响

目的观察联合转染反义DNMT1、DNMT3b基因真核表达载体对人胆管癌细胞株QBC-939的增殖能力和细胞凋亡的影响。方法分别构建反义DNMT1基因和反义DNMT3b基因真核表达载体,脂质体介导法将二者先后转染入QBC-939,经G418筛选和荧光细胞克隆挑选得到稳定转染细胞株,并用Western blot检测转染前后的蛋白表达变化;用MTT法和软琼脂克隆形成试验观察各组细胞的增殖能力,流式细胞术检测细胞生长周期及凋亡率的变化。结果Western blot检测证实转染反义基因能使相应蛋白表达水平降低;联合转染反义DNMT1、DNMT3b基因和单独转染反义DNMT1基因能影响QBC-939的生长曲线,使细胞增殖减慢,并以前者为甚;联合转染反义DNMT1、DNMT3b基因和单独转染反义DNMT1基因的细胞克隆形成率分别为(6.78±0.89)%和(14.86±2.13)%,明显低于未转染组;联合转染反义DNMT1、DNMT3b基因和单独转染反义DNMT1能影响QBC-939的细胞周期,使之阻滞于G1期,细胞凋亡率从(1.63±0.27)%分别增加到(18.47±1.46)%和(6.19±0.78)%;在上述效应检测中,单独转染反义DNMT3b基因实验组对QBC-939细胞的生长无明显影响。结论通过联合转染反义DNMT1和DNMT3b基因真核表达载体,能抑制胆管癌细胞的生长和增殖,并促进凋亡的发生,其效果明显优于单独转染DNMT1反义基因;在DNA甲基化的过程中,DNMT1起着主要的作用,DNMT3b扮演着协同的角色,二者通过甲基化途径对胆管癌的发生发挥作用。     

HMGA2与DNMT1在膀胱尿路上皮癌中的表达及临床意义

目的探讨HMGA2与DNMTl在膀胱尿路上皮癌中的表达变化及临床意义。方法利用免疫组化方法,以膀胱尿路上皮患者的癌组织及正常人的膀胱组织为研究对象,比较两组患者膀胱组织HMGA2与DNMTl的蛋白的表达水平变化。结果HMGA2与DNMTl在膀胱尿路上皮癌组织中的阳性表达率分别为65．00％和60．00％,与相对应的对照的正常膀胱组织相比较,HMGA2与DNMTl的表达增高显著,具有显著的统计学差异（P〈0．05）。结论HMGA2与DNMTl基因在膀胱尿路上皮癌的组织当中的过量表达,通过检测和干扰HM-GA2与DNMTl基因可能为膀胱尿路上皮的早期诊断和制定治疗方案提供新依据。     

罕见ICF综合征1例临床表型和分子遗传学分析

目的探讨免疫缺陷、着丝粒不稳定和面部异常(ICF)综合征的临床特征和遗传特点。方法回顾分析1例ICF综合征患儿的临床资料和基因检测结果。结果男性患儿,4岁,有反复感染;面部特征为圆脸、眼距稍宽、下颌尖、鼻梁稍扁、内眦赘皮明显、耳位低。免疫学检测示IgA缺如。基因测序发现患儿DNMT3B基因存在一个纯合错义突变c. 2506 GA,父母为杂合携带者,确诊为ICF综合征I型。结论 DNMT3B基因突变可引发ICF综合征,基因检测有助于提高此类罕见病的诊断。     

宫颈癌组织中DNA甲基转移酶1、3A和3B mRNA的表达

目的探讨DNA甲基转移酶1、3A和3B在宫颈癌发生发展中的作用。方法应用real-time PCR技术检测10例宫颈癌、10例宫颈上皮内瘤变和10例正常宫颈组织DNMT1、3A和3BmRNA的表达。结果宫颈癌组织、宫颈上皮内瘤变和正常宫颈中DNMT 1mRNA对GAPDH mRNA的相对丰度值分别是0.30±0.1、0.26±0.12和0.10±0.04,宫颈癌组织组织中与正常宫颈中DNMT 1表达有显著差异（P〈0.05）;宫颈癌组织、宫颈上皮内瘤变和正常宫颈中DNMT3A mRNA对GAPDH mRNA的相对丰度值分别是0.94±0.27、0.51±0.24和0.13±0.04,三组相比较有显著性差异（P〈0.05）;宫颈癌组织、宫颈上皮内瘤变和正常宫颈中DNMT 3B mRNA对GAPDH mR-NA的相对丰度值分别是0.30±0.09、0.28±0.11和0.11±0.04,宫颈癌组织组织中与正常宫颈中DNMT 3B表达有显著差异（P〈0.05）。结论 DNMT1、3A和3B参与了宫颈癌的发生,并在宫颈上皮内瘤变时就已经起了一定作用。     

Dysregulation of the epigenome in triple-negative breast cancers: Basal-like and claudin-low breast cancers express aberrant DNA hypermethylation

A subset of human breast cancer cell lines exhibits aberrant DNA hypermethylation that is characterized by hyperactivity of the DNA methyltransferase enzymes, overexpression of DNMT3b, and concurrent methylation-dependent silencing of numerous epigenetic biomarker genes. The objective of this study was to determine if this aberrant DNA hypermethylation (i) is found in primary breast cancers, (ii) is associated with specific breast cancer molecular subtypes, and (iii) influences patient outcomes. Analysis of epigenetic biomarker genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3) identified a gene expression signature characterized by reduced expression levels or loss of expression among a cohort of primary breast cancers. The breast cancers that express this gene expression signature are enriched for triple-negative subtypes — basal-like and claudin-low breast cancers. Methylation analysis of primary breast cancers showed extensive promoter hypermethylation of epigenetic biomarker genes among triple-negative breast cancers, compared to other breast cancer subclasses where promoter hypermethylation events were less frequent. Furthermore, triple-negative breast cancers either did not express or expressed significantly reduced levels of protein corresponding to methylation-sensitive biomarker gene products. Together, these findings suggest strongly that loss of epigenetic biomarker gene expression is frequently associated with gene promoter hypermethylation events. We propose that aberrant DNA hypermethylation is a common characteristic of triple-negative breast cancers and may represent a fundamental biological property of basal-like and claudin-low breast cancers. Kaplan–Meier analysis of relapse-free survival revealed a survival disadvantage for patients with breast cancers that exhibit aberrant DNA hypermethylation. Identification of this distinguishing trait among triple-negative breast cancers forms the basis for development of new rational therapies that target the epigenome in patients with basal-like and claudin-low breast cancers.     

DNA hypermethylation of SFRP2 influences the pathology of rheumatoid arthritis through the canonical Wnt signaling in model rats

Abstract

In this work, the expression of secreted frizzled related protein 2 (SFRP2) in rheumatoid arthritis (RA) model rats and the mechanisms of SFRP2 on the RA pathogenesis were investigated. Data suggested that SFRP2 was significantly down-regulated in RA model rats compared with normal control, and overexpression of SFRP2 suppressed the RA pathogenesis and the canonical Wnt signaling in fibroblast-like synovial cells (FLS) from RA model rats, whereas knockdown of SFRP2 got an opposite observation. Interestingly, 5-azadC treatment up-regulated the SFRP2 expression, inhibited the FLS proliferation, suppressed the expression of IL-6 and IL-8 and the fibronectin production, suggesting that the decreased SFRP2 in RA model rats was due to the DNA methylation. Furthermore, DNMT1 knockdown up-regulated the SFRP2 expression, DNMT1 overexpression inhibited the SFRP2, and the quantitative methylation-specific PCR (qMSP) confirmed that the DNMT1 has direct methylation roles for the SFRP2 promoter, leading to a regulation of FLS proliferation and fibronectin expression in RA model rats. In addition, up-regulated MeCP2 was involved in the SFRP2 regulation and the pathogenesis of RA model rats, and MeCP2 and DNMT1 have synergistic inhibition roles in the SFRP2 expression. Combination of DNMT1 and DNA methylation may be a promising treatment strategy for individuals with RA in which SFRP2 is down-regulated.     

Epigenetic enzymes: A role in aging and prospects for pharmacological targeting

The development of interventions aimed at improving healthspan is one of the priority tasks for the academic and public health authorities. It is also the main objective of a novel branch in biogerontological research, geroscience. According to the geroscience concept, targeting aging is an effective way to combat age-related disorders. Since aging is an exceptionally complex process, system-oriented integrated approaches seem most appropriate for such an interventional strategy. Given the high plasticity and adaptability of the epigenome, epigenome-targeted interventions appear highly promising in geroscience research. Pharmaceuticals targeted at mechanisms involved in epigenetic control of gene activity are actively developed and implemented to prevent and treat various aging-related conditions such as cardiometabolic, neurodegenerative, inflammatory disorders, and cancer. In this review, we describe the roles of epigenetic mechanisms in aging; characterize enzymes contributing to the regulation of epigenetic processes; particularly focus on epigenetic drugs, such as inhibitors of DNA methyltransferases and histone deacetylases that may potentially affect aging-associated diseases and longevity; and discuss possible caveats associated with the use of epigenetic drugs.     

DNMT1蛋白通过沉默MEG3基因促进视网膜母细胞瘤增殖

目的 探讨 DNMT1 蛋白是否通过沉默 MEG3 基因诱导视网膜母细胞瘤增殖。方法 通过转染 pcDNA-DNMT1 或si-DNMT1上调或干扰DNMT1的表达水平;通过转染pcDNA-MEG3或si-MEG3上调或干扰MEG3的表达水平;用Western blot检测视网膜母细胞瘤细胞系中DNMT1蛋白表达量;用CCK-8法及EdU法检测细胞的增殖能力;用实时荧光定量PCR技术检测转染后的视网膜母细胞瘤细胞中MEG3表达量的变化;干扰DNMT1表达后,用甲基化特异性PCR检测MEG3基因启动子DNA甲基化水平的变化。结果 视网膜母细胞瘤SO-RB50及HXO-RB44细胞中DNMT1蛋白表达量显著升高（P<0.05）。转染了 pcDNA-DNMT1 的 HXO-RB44 细胞中 DNMT1 蛋白表达增加,细胞增殖能力增加,MEG3 表达量降低;转染了 siRNADNMT1的SO-RB50细胞DNMT1蛋白表达减少,细胞增殖能力降低,MEG3表达量增加（P<0.05）。干扰DNMT1蛋白表达后,MEG3基因启动子DNA甲基化水平降低（P<0.05）。逆转DNMT1蛋白对MEG3基因的调控后,DNMT1蛋白调控RB细胞增殖的能力减弱（P<0.05）。结论 在视网膜母细胞瘤细胞中,DNMT1蛋白表达的上调,诱导了MEG3基因启动子DNA甲基化失活,最终导致细胞异常增殖。     

DNMT3b 基因在人膀胱癌组织中的表达及与临床预后的相关性

目的 探讨DNMT3b 在人膀胱癌组织的表达和意义及与膀胱癌患者术后生存期的相关性。方法 采用免疫组织化学技术（IHC）分别检测 DNMT3b 基因在 38 例手术切除人膀胱癌组织和38例相应癌旁组织的蛋白表达水平,分析其表达水平与患者年龄、性别、病理分级、肿瘤大小、T分期,淋巴转移和TNM分期临床病理参数的相关性,探讨DNMT3b对患者预后影响。结果 在38例膀胱癌组织中,DNMT3b 高表达率为 63.16%,而在对应的38例癌旁组织中高表达率为13.16%,两者间具有显著差异（P<0.05）。DNMT3b 在膀胱癌组织中高表达与患者病理分级（P=0.002）、肿瘤大小（P<0.001）、T分期（P<0.001）、淋巴转移（P=0.039）和TNM分期（P<0.001）相关,而与性别、年龄无明确相关性。多因素Logistic 回归分析发现,DNMT3b 的蛋白表达水平与肿瘤大小（P=0.008）和TNM 分级（P=0.042）相关。Kaplan-Meier生存分析显示,DNMT3b高表达组患者的总生存期明显缩短,与低表达组相比具有显著差异性（P=0.021）。结论 DNMT3b在膀胱癌组织中高表达,与病理分级、肿瘤大小、原发肿瘤范围、淋巴转移和临床分期等临床病理特征及总生存期密切相关,结果提示DNMT3b有望成为判断膀胱癌预后的标志物和肿瘤防治的新的靶点。     

Persistence of DNMT3A mutations at long‐term remission in adult patients with AML

Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1–50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B‐cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.