DNA甲基转移酶在妇科肿瘤中的研究进展

大量研究表明，许多种类的肿瘤细胞都有异常的DNA甲基化行为，抑癌基因常常被过量地甲基化而失去活性，而基因的DNA序列并不发生改变。DNA甲基化是由DNA甲基转移酶（DNMT）催化并维持的。DNMT通过调节细胞内甲基化过程而参与肿瘤的发生与发展，在有5′端调控区胞嘧啶．鸟嘌呤（CpG）岛甲基化异常参与的肿瘤细胞中常表现为过度表达，其活性增高是肿瘤细胞具有特征的早期分子改变。     

DNA甲基转移酶在肿瘤形成中的研究进展

DNA甲基化是基因表达调控中重要的调节方式之一，可通过影响癌基因和抑癌基因的表达以及基因组的稳定性而参与肿瘤形成。DNA甲基化是由DNA甲基转移酶（DNMT）催化发生并维持的，并认为DNMT活性增高是肿瘤细胞具有特征的早期分子改变，因而受到越来越多的学者关注。     

DNA甲基转移酶在肿瘤形成中的研究进展

DNA甲基化是基因表达调控中重要的调节方式之一,可通过影响癌基因和抑癌基因的表达以及基因组的稳定性而参与肿瘤形成。DNA甲基化是由DNA甲基转移酶(DNMT)催化发生并维持的,并认为DNMT活性增高是肿瘤细胞具有特征的早期分子改变,因而受到越来越多的学者关注。     

黄曲霉毒素B1诱导性大鼠肝癌超微结构观察及DNA甲基化转移酶3表达变化

由DNA甲基化转移酶(DNA methyltransferase,DNMT)活性改变诱导的DNA甲基化模式改变是肿瘤异常表观遗传修饰的重要机制,主要表现为基因组整体的低甲基化和区域性高甲基化,通过改变癌基因、抑癌基因的表达和基因组的稳定性,诱导正常细胞癌性转变[1-2].DNMT3基因是DNMT家系重要成员,在建立组织特异性甲基化模式方面发挥关键作用.黄曲霉毒素B1(aflatoxin B1,AFB1)为公认的原发性肝癌(hepatocellular carcinoma,HCC)致癌因素之一,其致癌过程中同样涉及DNA甲基化模式异常改变[3-6].DNMT3在肝细胞癌变过程中动态变化的研究少见报道,本研究旨在分析AFB1诱导性大鼠肝细胞癌变不同阶段DNMT3a mRNA和DNMT3b mRNA变化特征,初步研究AFB1诱导性大鼠HCC发生的表观遗传学机制.     

DNA甲基化与食管癌的关系

肿瘤细胞普遍存在DNA甲基化模式的改变,DNA甲基化异常包括原癌基因低甲基化和抑癌基因高甲基化。近年来研究结果表明,在食管癌发生过程中同样存在相关抑癌基因启动子区甲基化导致的基因表达的紊乱。其中由DNA甲基转移酶（DNA methyltransferase,DNMT）和去甲基化酶的活性改变导致的抑癌基因CpG岛超甲基化的研究,已成为食管癌发病机制研究中的热点之一。综述DNA甲基化的特点及其抑制基因转录和表达的分子生物学机制;DNA异常甲基化与食管癌发生发展的关系;食管癌相关肿瘤抑制基因的甲基化谱构成了食管癌独特的表遗传学标志;目前常用的甲基化检测手段及各方法的优缺点;DNA甲基化和去甲基化研究的展望及所需要解决的的问题等。     

肝癌患者p16基因甲基化与DNMTs表达相关性分析

目的:研究p16基因启动子区域异常甲基化所导致的基因表达异常在肝癌形成过程中的作用,探讨p16基因甲基化与DNMTs(DNMT1、DNMT2、DNMT3A和DNMT3B)表达之间的相关性。方法:检测肝癌患者癌组织、癌旁组织和肝硬化组织中p16基因的异常甲基化状态及p16、DNMTs基因mRNA的表达水平;采用甲基化特异性PCR技术检测甲基化状态,荧光定量技术检测mRNA的表达。结果:p16基因在肝癌组织、肝硬化组织和癌旁组织中的甲基化率分别为70.5%(31/44)、37.1%(13/35)和9.1%(4/44),肝癌组织和肝硬化组织中的甲基化改变与癌旁组织比较差异有统计学意义,P<0.01。31例甲基化阳性组织中17例p16基因表达降低或缺失,13例甲基化阴性组织中2例基因表达降低或缺失,甲基化阳性与阴性的p16基因表达水平存在明显差异。癌组织和肝硬化组织中4种DNMT mRNA水平均高于相应癌旁组织。其中DNMT1、DNMT3A和DNMT3BmRNA的表达水平差异有统计学意义,DNMT1:P值分别为0.009和0.020;DNMT3A:P值分别为0.005和0.010;DNMT3B:P值分别为0.039和0.036。DNMT2mRNA的表达水平虽然高于相应癌旁组织,但差异无统计学意义,P值分别为0.120和0.350。DNMT1、DNMT 3A和DNMT3B的表达与p16甲基化有相关性,P值分别为0.013、0.025和0.041。结论:p16基因甲基化是肝癌的早期、频发事件,其改变是其基因表达下降甚至失活的重要原因。DNMTs活性的改变可能促进p16基因的异常甲基化。     

DNA甲基化转移酶及DNA甲基化转移酶抑制剂在肿瘤研究中的新视角

表观遗传改变例如DNA甲基化涉及多种癌症的发生和进展。DNA甲基化包括可逆的甲基基团添加至CpG二核苷酸中5′位胞嘧啶上。而DNA甲基化转移酶(DNA methyltransferases,DNMT)是负责甲基基团添加至CpG二核苷酸的酶,与组蛋白修饰一起,是发生于转录抑制所必须的起始事件。已经证明在多种人恶性肿瘤中DNMT的表达增高,并且通过DNMT介导的基因失活促进肿瘤进展。DNMT在衰老与凋亡过程中都起到一定作用。表观遗传改变是潜在可逆的,这刺激DNA甲基化抑制剂药理学的发展,为肿瘤的治疗提供了一个新的途径。     

雷公藤内酯醇下调P53基因甲基化抑制肝癌SMMC-7721细胞的增殖

目的:研究雷公藤内酯醇(triptolide,TP)对人肝癌SMMC-7721细胞增殖的影响以及对P53基因的去甲基化作用。方法:MTT法检测TP对SMMC-7721细胞增殖的影响,甲基特异性PCR检测TP对SMMC-7721细胞P53基因甲基化的影响,RT-PCR检测SMMC-7721细胞甲基转移酶DNMT1、DNMT3a、DNMT3bmRNA的表达,Western blotting检测SMMC-7721细胞中P53蛋白的表达。结果:TP剂量依赖性抑制SMMC-7721细胞的增殖(P<0.05),40 ng/ml时的抑制率达(73.5±3.02)%,其半数抑制浓度(IC50)约为20 ng/ml。TP显著抑制SMMC-7721细胞中DNMT1、DNMT3a、DNMT3bmRNA的表达(P<0.05,P<0.01);TP作用后P53基因的高甲基化被逆转,并呈剂量依赖性;TP可显著增强SMMC-7721细胞中P53蛋白的表达。结论:TP可通过抑制甲基转移酶使P53基因去甲基化,促进P53蛋白的表达,从而抑制SMMC-7721细胞的增殖。     

雷公藤内酯醇下调P53基因甲基化抑制肝癌SMMC-7721细胞的增殖

目的:研究雷公藤内酯醇(triptolide,TP)对人肝癌SMMC-7721细胞增殖的影响以及对P53基因的去甲基化作用。方法:MTT法检测TP对SMMC-7721细胞增殖的影响,甲基特异性PCR检测TP对SMMC-7721细胞P53基因甲基化的影响,RT-PCR检测SMMC-7721细胞甲基转移酶DNMT1、DNMT3a、DNMT3bmRNA的表达,Western blotting检测SMMC-7721细胞中P53蛋白的表达。结果:TP剂量依赖性抑制SMMC-7721细胞的增殖(P<0.05),40 ng/ml时的抑制率达(73.5±3.02)%,其半数抑制浓度(IC50)约为20 ng/ml。TP显著抑制SMMC-7721细胞中DNMT1、DNMT3a、DNMT3bmRNA的表达(P<0.05,P<0.01);TP作用后P53基因的高甲基化被逆转,并呈剂量依赖性;TP可显著增强SMMC-7721细胞中P53蛋白的表达。结论:TP可通过抑制甲基转移酶使P53基因去甲基化,促进P53蛋白的表达,从而抑制SMMC-7721细胞的增殖。     

DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma

Aberrant DNA methylation and increased expression of DNA methyltransferases (DNMTs) are features of tumor cells. To investigate roles for DNMTs during hepatocarcinogenesis, we examined DNMT expression at both the mRNA and protein level in hepatocellular carcinomas (HCCs) and paired non-neoplastic liver tissues, along with measuring the DNA methylation status of five tumor suppressor genes. Expression of DNMT1, DNMT3a and DNMT3b mRNA was detected in 33.3, 59.3, and 55.6% of HCCs and 40.7, 22.2, and 0% of non-neoplastic liver tissues, respectively. DNMT1 and DNMT3a were immunoreactive in 100 and 48% of HCCs and 52 and 0% of non-neoplastic liver tissues. The DNMT3a mRNA expression profile showed significant correlation with its immunoreactivity (P=0.022). DNA methylation status of five tumor suppressor genes, HIC-1, p16, RASSF1A, p53, and RB1 was detected in 85.2, 48.1, 44.4, 22.2, and 0% of HCCs, respectively. There was no significant correlation between DNMT mRNA expression and DNA methylation (P>0.05). DNMT immunoreactivity was also not associated with DNA methylation except HIC-1 (P=0.036) and p53 methylation (P=0.009). Despite the lack of correlation between DNA methylation status and DNMT expression, the frequency of hypermethylation of tumor suppressor genes remained relatively high in HCCs, suggesting that regional DNA hypermethylation is involved in hepatocarcinogenesis and that there may be other mechanisms for increasing DNA methylation.     

DNA 甲基转移酶在肿瘤发病机制中的研究进展

DNA甲基化是表观遗传学的主要形式,而DNA甲基转移酶（ DNMTs）是DNA甲基化的主要调节酶,DNA甲基转移酶的激活参与了肿瘤的发生和发展过程,同时伴有肿瘤抑制基因的高甲基化沉默和低表达,是病人预后不良的标志;DNA甲基转移酶3b（ DNMT3b）的多态性及吸烟所致的DNMTs表达的改变是肿瘤发生的危险因素,靶向DNMTs治疗由于其细胞毒性小,是当前研究的一个热点。本文就DNA甲基转移酶在肿瘤发病机制中的作用做一综述。     

Alteration of DNA methyltransferases contributes to 5'CpG methylation and poor prognosis in lung cancer

Overexpression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers. However, physical binding of DNA methyltransferase (DNMT) to the hypermethylated promoter of tumor suppressor genes (TSGs) has never been demonstrated in tumor tissues. In addition, alteration of DNMT at the protein level has never been reported in the same series of cancer patients. By immunohistochemical analysis, we demonstrated that DNMT1, DNMT3a and DNMT3b proteins were highly expressed in a coordinate manner in lung tumors, particularly in smokers (P=0.037, by the Fisher exact test). Patients with DNMT1 overexpression had a trend of poorer prognosis than those without such overexpression, and this prognostic significance was apparent in squamous carcinoma (SQ) patients (P=0.041, by the log-rank test). Both DNMT1 and DNMT3b overexpressions correlated with hypermethylation in the TSG promoters, especially among smoking SQ patients (P=0.012). To further explore the molecular mechanisms between altered TSGs promoter methylation and overexpression of DNMTs protein, we performed a tissue chromatin-immunoprecipitation polymerase chain reaction assay for lung tumors and showed that the methylated FHIT, p16(INK4a) and RARbeta promoters were bound by both DNMT protein and methyl-CpG-binding protein 2. These data suggest that overexpression and strong binding of various DNMTs may result in promoter hypermethylation of multiple TSGs and ultimately lead to lung tumorigenesis and poor prognosis.     

DNA methylation and cancer

Tumor suppressor genes can be silenced by DNA methylation during cancer development. Aberrant DNA methylation is closely associated with histone deacetylases and histone methyltransferases that can modify histone amino-terminal lysines and develop specific histone codes, resulting in inactive chromatin formation. These processes change epigenetic information that builds up abnormal chromatin structure, and creates the unique features of cancer cells. It is well known that thousands of genes are deregulated in cancer cells. Epigenetic alterations involving aberrant DNA methylation is a possible mechanism that can explain the genome-wide abnormality of gene expression. The mechanism responsible for the aberrant DNA methylation is unclear now, however it seems that de novo DNA methyltransferases (DNMTs) play an important role in the process. DNMT3 A and DNMT3B are thought to be de novo DNMTs in human. A nucleoside analogue of cytidine induces demethylation of DNA in cancer cells by inhibiting the function of DNMTs. It is significant to elucidate precise mechanisms of aberrant DNA methylation and develop small molecules that can inhibit methylation.     

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition

It is observed that upregulation of DNMT3B enzyme in some cancers, including colon cancer, could lead to silencing of tumor suppressor genes. MiR-339 and miR-766 have been predicted to target 3′UTR of DNMT3B gene. Luciferase reporter assay validated that individual and co-transfection of miR-766 and miR-339 into the HEK293T cell reduced luciferase activity to 26% ± 0.41%, 43% ± 0.42 and 64% ± 0.52%, respectively, compared to the control (P < 0.05). Furthermore, transduction of miR-339 and miR-766 expressing viruses into colon cancer cell lines (SW480 and HCT116) decreased DNMT3B expression (1.5, 3-fold) and (3, 4-fold), respectively. In addition, DNA methylation of some tumor suppressor genes decreased. Expression of these genes such as SFRP1 (2 and 1.6-fold), SFRP2 (0.07 and 4-fold), WIF1 (0.05 and 4-fold), and DKK2 (2 and 4-fold) increased in SW-339 and SW-766 cell lines; besides, expression increments for these genes in HCT-339 and HCT-766 cell lines were (2.8, 4-fold), (0.005, 1.5-fold), (1.7 and 3-fold) and (0.04, 1.7-fold), respectively. Also, while in SW-766, cell proliferation reduced to 2.8% and 21.7% after 24 and 48 hours, respectively, SW-339 showed no reduced proliferation. Meanwhile, HCT-766 and HCT-339 showed (3.5%, 12.8%) and (18.8%, 33.9%) reduced proliferation after 24 and 48 hours, respectively. Finally, targeting DNMT3B by these miRs, decreased methylation of tumor suppressor genes such as SFRP1, SFRP2, WIF1 and DKK2 in the mentioned cell lines, and returned the expression of these tumor suppressor genes which can contribute to lethal effect on colon cancer cells and reducing tumorigenicity of these cells.     

Effect of Zebularine in Comparison to and in Combination with Trichostatin A on CIP/KIP Family (p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2), DNMTs (DNMT1, DNMT3a,and DNMT3b), Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) Gene Expression,Cell Growth Inhibition and Apoptosis Induction in Colon Cancer LS 174T Cell Line

Background: A pattern of epigenetic modifications and changes, DNA methylation and histone modification, is central to many human cancers. A variety of tumor suppressor genes (TSGs) have been demonstrated to be silenced because of histone deacetylation and DNA hypermethylation in several cancers. Recent in vitro studies have shown that two known mechanisms of epigenetic alteration consisting of methylation and histone deacetylation seem to be the best candidate mechanisms for inactivation of CIP/KIP family (p21Cip1/Waf1/Sdi1, and p27Kip1) in numerous cancers. Numerous investigations have indicated that DNA demethylating and histone deacetylase inhibitors (HDACIs) can restore the CIP/KIP family gene expression. Previously, we evaluated the effect of trichostatin A (TSA) and 5-aza-2′-deoxycytidine (5-AZA-CdR) on hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of zebularine in comparison to and in combination with trichostatin A on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNMT1, DNMT3a and DNMT3b, Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) gene expression, cell growth inhibition and apoptosis induction in colon cancer LS 174T cell line. Materials and Methods: The colon cancer LS 174T cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and trichostatin A can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.