多能性胚胎干细胞与DNA甲基化的关系

胚胎干细胞是一种能够维持自我更新、具有无限扩增能力的多能性干细胞。灵长类多能干细胞（iPSCs）根据其发育能力、细胞形态、基因表达谱以及表观遗传学的差异分为初始态多能干细胞（pPSCs）和原始态多能干细胞（nPSCs）。nPSCs因其容易进行基因工程处理以及体内外再生出功能组织器官等优势而在临床潜在应用上备受关注,因而有效维持ESCs的原始状态对其用于基础及临床研究具有重要意义。nPSCs的线粒体活性和自我更新能力高于pPSCs,且这两种多能性干细胞在DNA甲基化等方面都存在明显差别,DNA甲基化在nPSCs的转化及代谢中起到重要的作用。本文综述了DNA甲基化对ESCs的作用,特别是维持原始态的作用。     

HaCaT细胞中FUT4表达与其启动子区甲基化的相关性分析

岩藻糖基转移酶Ⅳ(Fucosyltransferase Ⅳ,FUT4)在正常细胞中表达量很低,但其低表达的调控机制以及是否受其启动子甲基化调控并不十分清楚。文章采用Western blot、免疫荧光和Real-time PCR的方法检测正常人永生化表皮细胞系HaCaT细胞FUT4的表达,观察DNA甲基转移酶抑制剂5-aza-dC处理对FUT4表达的影响。应用甲基化特异性PCR方法分析HaCaT细胞中FUT4启动子甲基化状态。结果表明,HaCaT细胞中FUT4的表达水平明显低于人表皮鳞癌细胞A431和SCC12。5 μmol/L的5-aza-dC处理72 h的HaCaT细胞,其FUT4 mRNA水平明显升高,并且与未经5-aza-dC处理的对照组相比,U引物扩增检测到的产物量增加,M 引物扩增检测到的产物量明显减少。这些结果表明,HaCaT细胞中FUT4的低表达可能与其启动子区CpG岛甲基化有关。     

胚胎发育过程中的DNA甲基化作用

哺乳动物的正常发育取决于表观遗传学调控机制准确无误地运行.其中尤为重要的是发生在原生殖细胞和胚胎中的基因组范围内的DNA甲基化模式重排等表观遗传学修饰.胚胎发育过程中的DNA甲基化作用与基因印记的建立、基因表达的调控以及细胞和胚胎的形态建成都密切相关.DNA甲基化发生机制和功能的阐明将对哺乳动物个体发育与人类疾病研究有重要意义.     

Oct4基因在猪孤雌激活和体外受精早期胚胎中的表达特征

目的研究植入前胚胎发育重要基因Oct4在猪孤雌和体外受精胚胎中的表达特征。方法收集成熟卵母细胞、孤雌和体外受精2细胞、4细胞、8细胞胚胎和囊胚,做荧光即时定量PCR检测,以体外成熟的猪卵母细胞做对照分析相对表达量。结果孤雌组和体外受精组胚胎在8细胞期Oct4表达量均最高(P<0.05),在孤雌和体外受精组囊胚相对于其他时期Oct4表达量最低(P<0.05)。在同一时期孤雌和体外受精胚胎上Oct4表达并没有差异。结论多能性基因Oct4在卵裂发育时期表达量动态变化,孤雌胚胎在一定程度上可作为体外胚胎基因表达的模型,且不同的胚胎培养条件可能导致基因表达的差异。     

应用亚硫酸氢盐修饰后PCR联合TA克隆测序对E-cadherin启动子区甲基化水平的检测

E-cadherin是一种细胞粘附因子,通过增强细胞之间的粘附而起到抑制肿瘤转移的作用.Ecadherin基因启动子区的高甲基化是导致其在众多肿瘤细胞中表达下调甚至缺失的主要原因之一.本实验首先抽提SGC-7901细胞(胃腺癌细胞)、A549细胞(肺腺癌细胞)、MCF-7细胞(乳腺癌细胞)等3个肿瘤细胞株的全基因组DNA,然后对抽提的DNA进行亚硫酸氢盐修饰和纯化回收,根据修饰后的DNA序列设计引物并对其进行PCR扩增.然后将PCR扩增产物与pUC-T TA载体连接并转化入感受态大肠杆菌DH5α中进行培养,对筛选出的含有阳性重组子的菌落进行测序.测序结果显示,3个肿瘤细胞株的E-cadherin基因启动子区的CpG岛都呈现了高度的甲基化,亚硫酸氢盐的修饰效率达到了99.2%.综上研究表明,亚硫酸氢盐修饰后PCR(BSP)联合TA克隆测序可以对肿瘤细胞某基因启动子区CpG岛的甲基化水平进行精确量化,研究所使用的3个肿瘤细胞株均可作为研究肿瘤细胞E-cadherin基因甲基化的细胞模型.     

5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制

目的: 探讨小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)向生殖细胞(Embryonic germ cells,EG)分化过程中5-杂氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC) 对DNA甲基化转移酶Dnmt1和Dnmt3a及生殖细胞特征基因Mvh表达变化的DNA甲基化调控机制。方法:将mES细胞分化形成拟胚体(embryoid bodies, EBs) 作为向生殖细胞分化的启动步骤,采用不同浓度(0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,3μmol/L)处理EBs,RT-PCR实时荧光定量RT-PCR和Western blot分别检测检测在5-Aza-dC处理前后Dnmt1和Dnmt3a在ES细胞和EBs中的表达,甲基化特异性PCR(MSP)检测原始生殖细胞分化特征基因Mvh启动子甲基化状态。结果: 5-Aza-dC的浓度在0.05 μmol/L~1 μmol/L之间时,EBs保持较高的存活率而EBs的形态明显发生了变化;5-Aza-dC 处理后, Dnmt1和Dnmt3a在EBs中mRNA表达量明显降低,其变化特点与WB结果相一致。MSP和测序结果显示, Mvh启动子区表现为部分甲基化,5-Aza-dC 处理后的4d EBs中Mvh CpG岛有4个CG位点发生突变,而mES细胞中未见突变。结论: EBs经5-Aza-dC处理后,Dnmt1和Dnmt3a的表达明显下调;同时,Mvh启动子发生部分甲基化,有可能启动了向生殖细胞的分化进程。     

NDRG2基因启动子甲基化与肺腺癌细胞中mRNA表达相关性的实验研究

目的:探讨肺腺癌细胞中NDRG2基因启动子甲基化状态及其与基因表达的关系。方法:甲基化焦磷酸测序技术检测启动子区域甲基化状态,荧光定量PCR技术检测不同药物浓度下培养细胞中NDRG2基因mRNA的表达水平,分析启动子区域甲基化与基因表达之间的关系。结果:在体外培养细胞中检测到NDRG2基因启动子区域呈现不同程度的甲基化,甲基化频率分别为肺癌A549细胞71.8%、GLC-82细胞86.1%、人脐静脉内皮ECV-304细胞36.8%、胃上皮GES-1细胞42.9%。NDRG2基因mRNA表达与其启动子甲基化程度成反比,甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-Aza-CdR)作用于细胞后,A549和GLC-82细胞中NDRG2基因的mRNA转录明显上调,至72 h差异显著(P0.05)。结论:肺腺癌细胞中NDRG2基因启动子CpG岛存在高甲基化,甲基化程度与该基因的表达具有负相关性,5-Aza-CdR能在一定程度上提高NDRG2的转录水平。     

Zfx基因与干细胞自我更新

干细胞具有自我更新保持不分化状态的特性,不同的干细胞具有不同的自我更新机制. Zfx基因(zinc fin ger-X gene)在部分胚胎干细胞和造血干细胞中高表达,该基因高表达有利于胚胎干细胞和造血干细胞自我更新; Zfx基因表达不足或缺乏的胚胎干细胞和造血干细胞自我更新的能力下降,细胞凋亡明显增加.在胚胎干细胞和造血干细胞中发现一些Zfx基因直接调控的靶基因,Zfx 基因可能是控制各种干细胞自我更新的共同的分子机制. Zfx基因表达不足不影响胚胎干细胞和造血干细胞的分化,缺乏 Zfx基因的胚胎干细胞和造血干细胞能够正常分化为各自的功能细胞.     

MicroRNAs与胚胎干细胞研究

MicroRNAs(miRNAs)是一种大小约20～25个碱基的非编码小分子RNA,一般通过特异性抑制靶蛋白翻译或降解靶基因mRNA发挥负调控基因表达的作用.胚胎干细胞(embryonic stem cells,ES细胞)是从植入前早期胚胎内细胞团或原始生殖细胞中分离得到并能在体外长期培养的高度未分化的多能细胞系,在揭示胚胎早期发育机理、药物筛选、临床再生医学等领域具有广泛的应用前景.最近研究发现miRNAs在ES细胞自我更新和分化过程中均发挥着重要的调控作用,但具体调控机制尚未完全阐明.进一步深入研究miRNAs在ES细胞中的作用,全面了解ES细胞自我更新和定向分化的机制是实现ES细胞广阔临床应用前景的基础.     

NANOGP8基因在肺癌细胞系中甲基化水平与表达研究

目的:研究NANOGP8基因在肺癌细胞系A549中启动子区域甲基化水平及其与基因表达的相关性。方法:利用MethPrimer甲基化岛预测和甲基化引物设计软件,预测NANOGP8启动子区甲基化位点。分别从人成纤维细胞系和肺癌细胞系中提取基因组DNA,经过亚硫酸氢钠处理后,用针对甲基化位点设计的引物进行PCR扩增,获得相应区段DNA后,连接到pGEM-T载体,转化大肠杆菌鉴定阳性克隆后,测序并与GenBank数据库中NANOGP8基因组DNA序列比对,获得其甲基化水平数据。分别提取两个细胞系的总RNA,RT-PCR获得相应cDNA进行PCR扩增,扩增产物经琼脂糖凝胶电泳后,经酶切和测序验证,获取其表达水平数据。结果:成功预测NANOGP8的两个区域有甲基化位点,并检测到人成纤维细胞系和肺癌A549细胞系中NANOGP8启动子甲基化水平分别为59.7%和12.5%,表达检测结果显示在A549细胞系中检测到NANOGP8的基因片段,而在人成纤维细胞系中没有扩增到相应产物。结论:在正常成体细胞中NANOGP8基因由于启动子的高度甲基化而沉默,而在肺癌细胞系中NANOGP8基因启动子去甲基化激活其表达。NANOGP8基因的表达与其启动子区域去甲基化密切相关,同时NANOGP8在肺癌细胞分化过程中发挥重要作用。     

Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6xDBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.     

Epigenetic mechanisms are behind the regulation of the key genes associated with the osteoblastic differentiation of the mesenchymal stem cells: The role of zoledronic acid on tuning the epigenetic changes

Mesenchymal stem cells (MSCs) are multipotent stem cells and show distinct features such as capability for self-renewal and differentiation into several lineages of cells including osteoblasts, chondrocytes, and adipocytes. In this study, the methylation status of the promoter region of zinc finger and BTB domain containing 16 (ZBTB16), twist-related protein 1(Twist1), de novo DNA methyltransferases 3A (DNMT3A), SRY-box 9 (Sox9), osteocalcin (OCN), and peroxisome proliferator-activated receptor γ2 (PPARγ2) genes and their messenger RNA (mRNA) expression levels were evaluated during the osteoblastic differentiation of MSCs (ODMSCs). We planned two experimental groups including zoledronic acid (ZA)-treated and nontreated cells (negative control) which both were differentiated into the osteoblasts. Methylation level of DNA in the promoter regions was assayed by methylation-specific-quantitative polymerase chain reaction (MS-qPCR), and mRNA levels of the target inhibitory/stimulatory genes during osteoblastic differentiation of MSCs were measured using real-time PCR. During the experimental induction of ODMSCs, the mRNA expression of the OCN gene was upregulated and methylation level of its promoter region was decreased. Moreover, Sox9 and PPARγ2 mRNA levels were attenuated and their promoter regions methylation levels were significantly augmented. However, the mRNA expression of the DNMT3A was not affected during the ODMSCs though its methylation rate was increased. In addition, ZA could enhance the expression of the ZBTB16 and decrease its promoter regions methylation and on the opposite side, it diminished mRNA expression of Sox9, Twist1, and PPARγ2 genes and increased their methylation rates. Intriguingly, ZA did not show a significant impact on gene expression and methylation levels the OCN and DNMT3A. We found that methylation of the promoter regions of Sox9, OCN, and PPARγ2 genes might be one of the main mechanisms adjusting the genes expression during the ODMSCs. Furthermore, we noticed that ZA can accelerate the MSCs differentiation to the osteoblast cells via two regulatory processes; suppression of osteoblastic differentiation inhibitor genes including Sox9, Twist1, and PPARγ2, and through promotion of the ZBTB16 expression.     

RG108对肺腺癌A549细胞增殖、凋亡及RASSF1A基因表达的影响

目的探讨DNA甲基转移酶抑制剂RG108对人肺腺癌细胞株A549增殖、凋亡以及对RASSF1A（Ras as-sociation domain proteinfamily1）基因启动子区域甲基化状态、表达的影响。方法用20μmol/L的RG108对A549细胞进行化学干预72h,用MTT法检测细胞生长抑制率;流式细胞术检测细胞周期以及凋亡情况;RT-PCR观察RASSF1A基因mRNA水平变化;Western blot检测RASSF1A蛋白的表达;甲基化特异性PCR（MS-PCR）检测RASSF1A基因启动子区域甲基化状态的改变。结果经RG108干预72h后,A549细胞的抑制率为17.2±0.43%,细胞周期阻滞于G0/G1期,并引起细胞凋亡。RT-PCR和Western blot结果显示在干预组细胞中分别出现RASSF1A基因的DNA条带（329bp）和蛋白质条带（39kD）,而对照组中无相应条带出现。RASSF1A基因启动子区域由甲基化状态转变为非甲基化状态。结论RG108可使RASSF1A基因启动子区域去甲基化,并通过该机制诱导RASSF1A基因在人肺腺癌细胞株A549中表达。     

Effect of TET1 regulating MGMT on chemotherapy resistance of oral squamous cell carcinoma stem cells

The study was to evaluate the effect of ten‐eleven translocation 1 (TET1) regulating o6‐methylguanine‐DNA methyltransferase (MGMT) in chemotherapy resistance of oral squamous cell carcinoma (OSCC) stem cells. OSCC stem cells were divided into the blank, negative control (NC), TET1‐siRNA, TET1‐siRNA + MGMT‐OE, and MGMT‐OE groups. Methylation‐specific polymerase chain reaction (MSP), qRT‐PCR and Western blotting were conducted to detect the methylation status of MGMT, expressions of TET1, MGMT, ABCG2, and Oct‐4. Cell proliferation, cisplatin chemosensitivity, and cell cycle and apoptosis, were detected using CCK8 and flow cytometry. A chromatin immunoprecipitation (ChIP) assay was employed for detecting the link between TET1 and MGMT gene promoters. In comparison to the NC group, the TET1‐siRNA group exhibited increased levels of MGMT methylation, the number of apoptotic cells and cisplatin chemosensitivity consisting of varying concentrations, however, decreased levels of mRNA and protein expressions of TET1 as well as MGMT, cell viability, the number of cells in the S phase, and protein expressions of ABCG2 and Oct‐4 were all have diminished amounts. The TET1‐siRNA + MGMT‐OE and MGMT‐OE groups had higher MGMT mRNA and protein expression, as well as increased protein expressions of ABCG2 and Oct‐4, greater cell activity, higher number of cells in the S phase, decreased apoptotic rates in cells and decreased cisplatin chemosensitivity with different concentrations. Our study provided evidence that low‐expression of TET1 in OSCC stem cells may stimulate MGMT promoter methylation, while inhibiting MGMT mRNA expression, this ultimately strengthens the sensitivity of OSCC stem cells in regards to chemotherapeutics.