5-氮-2'-脱氧胞嘧啶对U266细胞系P16基因去甲基化作用研究

  目的 探讨5-氮-2'-脱氧胞嘧啶（5-Aza-CdR）诱导骨髓瘤细胞系U266 p16基因 DNA 5′CpG岛去甲基化作用及对U266细胞增生的影响。方法 采用巢式甲基特异性PCR法（n-MSP）、DNA序列分析、RT-PCR、细胞生长曲线、流式细胞仪DNA含量分析法检测5-Aza-CdR对U266细胞 p16基因去甲基化作用及其对U266细胞的生长、增生及细胞周期的影响。结果 （1）5-Aza-CdR能够逆转U266细胞 p16 基因异常甲基化；（2）5-Aza-CdR能激活p16基因沉默的再转录；（3）5-Aza-CdR能下调U266细胞甲基转移酶DNMT1、DNMT3A、DNMT3B 的表达并呈浓度依赖性；（4）5-Aza-CdR作用的U266细胞被阻滞于G0 ~ G1期。结论 5-Aza-CdR可能通过抑制甲基转移酶直接对p16 基因去甲基化，逆转U266 细胞DNA 异常甲基化，并有效地激活因高甲基化所致p16基因沉默的再转录     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.     

5 Aza CdR通过DNMT1调控卵巢癌细胞ERCC1基因甲基化及其表达

目的:研究5-氮杂-2'-脱氧胞苷(5-Aza-CdR)在卵巢癌细胞系SKOV3中对核苷酸切除交叉修复互补基因1(exeision repair cross complementation group 1,ERCC1)表达的影响及可能的机制.方法:设计特异性针对DNA甲基转移酶1(DNAmethyhransferase 1,DNMT1)基因的shRNA转染人人卵巢癌细胞系SKOV3细胞中,Western blotting检测SKOV3细胞DNMT1以及ERCC1的表达变化;利用不同浓度5-Aza-CdR于不同时间点处理卵巢癌SKOV3细胞,Western blotting检测DNMTI和ERCC1蛋白在处理前后的变化,利用亚硫酸氢钠法检测ERCC1基因启动子区域甲基化水平.结果:0.5、1.0、2.0、4.0μmol/L的5-Aza-CdR作用于SKOV3细胞后,DNMT1表达水平呈浓度依赖性降低,而ERCC1表达水平呈浓度依赖性升高;使用终浓度为1.0 μmol/L的5-Aza-CdR处理SKOV3细胞12、24、36 h后,DNMT1表达水平呈时间依赖性降低,而ERCC1表达水平呈时间依赖性升高,亚硫酸氢钠法检测示药物处理前ERCC1启动子区域处于高甲基化水平,在用1.0μmol/L的5-Aza-CdR处理后,其启动子发生了去甲基化.结论:5-Aza-CdR通过DNMT1调控卵巢癌SKOV3细胞中ERCC1基因的甲基化及其表达水平.     

G6PD缺陷对 1,4-苯醌致K562细胞DNA甲基化的影响及其机制

目的：研究G6PD缺陷对1,4-苯醌（1,4-BQ）致K562细胞DNA甲基化的影响及其机制。方法：分别采用10和20 μmol/L的1,4-BQ溶液对G6PD缺陷K562细胞和正常表达细胞进行染毒,以未染毒组为对照,各组均设置12、24、48 h共3个染毒时间,另在20 μmol/L 1,4-BQ染毒组细胞中加入1.5 mmol/L谷胱甘肽（GSH）进行干预。采用比色法检测全基因组DNA甲基化相对水平,qPCR法检测甲基转移酶DNMT1、DNMT3a、DNMT3b mRNA表达水平,Western blot法检测DNMT1、DNMT3a蛋白表达水平。结果：10和20 μmol/L 1,4-BQ染毒后的K562细胞全基因组DNA甲基化水平升高（P<0.05）,除20 μmol/L 1,4-BQ染毒48 h组外,其他各染毒组的G6PD缺陷K562细胞全基因组DNA甲基化水平均高于正常细胞（P<0.05）。在1,4-BQ染毒后,G6PD缺陷细胞的DNMT1、DNMT3a、DNMT3b mRNA水平以及DNMT1、DNMT3a蛋白表达水平均高于正常细胞（P<0.05）。加入GSH后,G6PD缺陷细胞和正常细胞的全基因组DNA甲基化相对水平、DNMTs mRNA及蛋白表达水平的差异的无统计学意义（P均>0.05）。结论：G6PD缺陷可能以抑制K562细胞GSH合成的方式增加氧化应激水平,最终导致细胞DNMTs生成的增多以及全基因组DNA甲基化程度的升高。     

5-氮杂-2＇-脱氧胞苷对人食管癌细胞株EC1化疗敏感性的影响

目的探讨去甲基化药物5-氮杂-2＇-脱氧胞苷（5-aza-2＇-deoxycytidines,5-Aza-CdR）对食管癌细胞化疗敏感性的影响及其机制。方法经5-Aza-CdR处理人食管癌细胞株EC1后,采用MTT检测各组细胞的化疗敏感性,流式细胞仪分析各组细胞的细胞周期变化,Western blot测定对甲基化转移酶（DNA methyltransferase,DNMT）表达的影响。结果经5-Aza-CdR处理后,EC1细胞对顺铂的化疗敏感性提高。5-Aza-CdR可诱导EC1细胞出现明显的G0/G1期阻滞。DNMT3B在食管癌细胞EC1中表达明显增高,经5-Aza-CdR作用后,其表达水平显著降低。结论 5-Aza-CdR可提高人食管癌EC1细胞对顺铂的敏感性,将细胞阻滞于G0/G1期并下调DNMT3B表达可能是其重要的作用机制。     

EGFR信号通路相关的miR-125a-5p调控肺癌转移的研究

[目的]了解表皮生长因子受体（EGFR）信号通路和microRNA（miRNA）表达的关系,探讨miRNA在肺癌转移中的作用。[方法]采用miRNA芯片分析表皮生长因子（EGF）刺激前后肺癌细胞中miRNA表达谱的改变;利用Transwell Insert迁移与侵袭实验,体外划痕愈合实验了解miR-125a-5p抑制肺癌细胞迁移与侵袭的能力;微管形成实验了解脐静脉血管内皮细胞（ECV304）体外形成血管的能力。[结果]芯片检测与RT-PCR显示34个miRNA的表达受到EGFR信号通路活化的影响,其中miR-125a-5p改变最明显;转染miR-125a-5p抑制物后PC9细胞的迁移数（286.67±10.51 vs 218.00±9.54,P=0.001）,侵袭数（245.67±5.51 vs 186.33±5.03,P〈0.001）,创伤愈合能力（P〈0.05）较阴性对照增加,ECV304细胞形成的微管数量也增多。[结论]miR-125a-5p可能受EGFR信号通路调节,并且对肺癌细胞具有转移抑制作用。     

miR-125a-3p靶向负调控PLK4抑制神经母细胞瘤细胞增殖及其机制探讨

目的:研究miR-125a-3p对神经母细胞瘤细胞增殖的影响,并探讨其作用机制。方法:运用qRT-PCR法检测Hela、SH-SY-5Y、SHEP、SK-N-BE细胞中miR-125a-3p的表达;将miR-125a-3p组（转染miR-125a-3p mimics）、miR-NC组（未转染细胞）、inhibitor-NC组（转染空inhibitor）、miR-125a-3p inhibitor组（转染miR-125a-3p inhibitor）、siPLK4组（转染siPLK4）、miR-125a-3p+Vector组（miR-125a-3p mimics和pcDNA 3.1共转染）、miR-125a-3p+PLK4组（miR-125a-3p mimics和pcDNA 3.1-PLK4共转染）以脂质体法转染至SH-SY-5Y细胞;MTT法检测各组细胞的增殖情况;Western blot检测各组细胞中PLK4、PIK3CA、Akt、p-Akt蛋白的表达;双荧光素酶报告基因检测实验检测各组细胞的荧光活性。结果:与Hela细胞相比,SH-SY-5Y、SHEP、SK-N-BE细胞中miR-125a-3p的表达均显著降低（P<0.05）;与Control组相比,miR-125a-3p组细胞的增殖显著降低,PLK4、PIK3CA、p-Akt蛋白的表达量均显著降低（P<0.05）;PLK4为miR-125a-3p的靶基因。过表达PLK4可逆转miR-125a-3p对SH-SY-5Y细胞增殖及PI3K/Akt信号通路的抑制作用。结论:miR-125a-3p可抑制神经母细胞瘤细胞增殖,其作用机制与靶向负调控PLK4有关,将可为神经母细胞瘤的治疗提供新靶点。     

5-氮杂-2ˊ-脱氧胞苷对乳腺癌细胞RASSF1A甲基化的逆转作用

目的：通过甲基化抑制剂5-氮杂-2ˊ-脱氧胞苷（5-Aza-CdR）作用于乳腺癌细胞株MCF-7,探索5-Aza-CdR逆转乳腺癌抑癌基因RASSF1A甲基化作用的机理。方法：体外培养乳腺癌细胞株MCF-7细胞,使用不同浓度5-氮杂-2ˊ-脱氧胞苷处理MCF-7细胞,对照组不加药。MSP及Western blot分别检测经5-Aza-CdR处理前、后RASSF1A DNA、蛋白的表达情况。结果：经去甲基化药5-Aza-CdR作用后,实验组恢复了RASSF1A的表达,明显高于对照组（ P〈0.05）。结论：去甲基化药5-Aza-CdR可以逆转乳腺癌细胞RASSF1A的甲基化作用,恢复了RASSF1A蛋白的表达,为临床治疗乳腺癌提供了理论基础。     

血浆miRNA在肺癌早期筛查中作用的病例对照研究

目的:探讨miR-122-5p等miRNA在早期肺癌患者血浆中的表达水平及其与肺癌发病风险的关联性。方法:运用高通量测序技术筛选在早期肺癌患者和对照者血浆中差异表达的miRNA,通过实时荧光定量PCR（quantitative real-time PCR,qRT-PCR)检测miRNA的表达。采用性别、年龄±5岁进行1∶2匹配的病例对照研究,使用条件Logistic回归分析miRNA与肺癌发病风险的关联。结果:早期肺癌组血浆miR-122-5p、miR-199a-5p、miR-221-3p、miR-27b-3p和miR-99a-5p的表达水平均显著高于对照组（P＜0.01）;用于检测早期肺癌时,miR-199a-5p的灵敏度可达95.8%,miR-99a-5p的特异度可达81.2%;血浆miR-122-5p、miR-199a-5p、miR-221-3p、miR-27b-3p和miR-99a-5p的表达与肺癌发病风险升高存在显著关联,第四分位数组的肺癌发病风险较高,分别是第一分位数组的9.613倍（95%CI:1.488~62.118,Ptrend=0.011）、23.863倍（95%CI:2.208~257.938,Ptrend=0.007）、27.416倍（95%CI:2.575~291.919,Ptrend=0.003）、23.626倍（95%CI:2.044~273.070,Ptrend=0.005）和72.504倍（95%CI:3.519~1 493.742,Ptrend=0.004）。结论:miR-122-5p、miR-199a-5p、miR-221-3p、miR-27b-3p和miR-99a-5p是肺癌发病的危险因素。     

miR-32-5p通过靶向Dickkopf相关蛋白3的表达调控乳腺癌MDA-MB-231 细胞的生物学行为

目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因,干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA,ENCORI数据库验证差异miRNA的表达,以选定最显著的差异表达 miRNA 为研究对象;利用 Starbase、miRDB 和 miRWalk 数据库预测 miR-32-5p 的靶基因,利用DAVID数据库对靶基因进行GO分析和KEGG分析,利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选,从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列,分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA,ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致,故选择其进行研究;预测得到198个miR-32-5p 潜在的靶基因并鉴定出 10 个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12）,其中DKK3的度值最大可能在乳腺癌中较为重要,于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01）,其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比,过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01）,敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路,过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。     

Methylation of miR-124a-1, miR-124a-2, and miR-124a-3 genes correlates with aggressive and advanced breast cancer disease

Aberrant DNA methylation on CpG islands is one of the most consistent epigenetic changes in human cancers, and the process of methylation is catalyzed by the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. Recent reports demonstrate that deregulation of miR-124a, one of the frequently methylated microRNAs in human cancers, is related to carcinogenesis. The aim of this study was to evaluate the frequencies of methylation of the three genomic loci encoding the miR-124a in primary breast cancers and to investigate their relationships with the clinicopathological characteristics of the tumors and with the expression levels of DNMT1, DNMT3a, and DNMT3b. The methylation status of the three genomic loci encoding the miR-124a (miR-124a-1, miR-124a-2, and miR-124a-3) was analyzed in fresh-frozen tumor samples using methylation-specific PCR in a large series of invasive breast ductal carcinomas (n?=?60). Results were correlated to several clinicopathological characteristics of the tumors and to the expression levels of DNMT1, DNMT3a, and DNMT3b, determined by immunohistochemistry. Promoter hypermethylation of miR-124a-1, miR-124a-2, and miR-124a-3 was detected in 53.3, 70, and 36.7 % of cases, respectively. Methylation of miR-124a-2 correlated to patients with age higher than 45 years (P?=?0.008) and to postmenopausal patients (P?=?0.03), whereas methylation of miR-124a-3 correlated significantly to tumor size >20 mm (P?=?0.03). Interestingly, simultaneous methylation of the three genes encoding miR-124a correlated significantly with the presence of lymph node metastasis (P?=?0.01) and high mitotic score (P?=?0.03). No significant correlation was found between promoter hypermethylation of miR-124a and expression of hormone receptors or HER2/neu. With regard to DNMT expression, no correlation was found between DNMT1 or DNMT3a expression and promoter methylation of any tested microRNA. However, DNMT3b overexpression correlates significantly with the hypermethylation of miR-124a-3 (P?=?0.03). Our data indicates that miR-124a-1, miR-124a-2, and miR-124a-3 genes are frequently methylated in breast cancer and play a role in tumor growth and aggressivity.     

DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

The purpose of the present study was to examine the epigenetic mechanism by which microRNA (miR)-152-3p regulates proliferation in non-small cell lung cancer A549 cells via neural cell adhesion molecule 1 (NCAM1). Bisulfite sequencing PCR (BSP), the gold standard for methylation detection, uses bisulfite-treated DNA to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. It was conducted and demonstrated a relatively high level of methylation in the miR-152-3p promoter region. Chromatin immunoprecipitation was combined with PCR to detect the binding of DNA methyltransferase 3B (DNMT3B) protein to miR-152-3p, which tends to occur in the core region of the miR-152-3p gene in A549 cells. Luciferase assay identified NCAM1 as the target gene of miR-152-3p. MTT, colony formation and Transwell assays indicated that miR-152-3p could decrease cell proliferation and invasion and in addition to reducing the expression level of NCAM1. Overexpression of NCAM1 could attenuate the effect of miR-152-3p. DNMT3B knockdown decreased the proliferative ability of A549 cells and increased the expression of miR-152-3p, while decreased that of NCAM1. After treatment with miR-152-3p inhibitor, these effects were attenuated and the NCAM1 expression level was upregulated. The results indicated that miR-152-3p may suppress the proliferation of A549 cells by downregulating NCAM1. In addition, DNMT3B negatively regulated the expression of miR-152-3p via modulation of the methylation level in the miR-152-3p core region, thus mediating the proliferation of lung tumor cells.     

DNA methylation regulates MicroRNA expression

MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.