A differentially methylated region of the DAZ1 gene in spermatic and somatic cells

Aim:To investigate the methylation status of the deleted in azoospermia 1(DAZ1)gene promoter region in differentcell types.Methods:Using CpG island Searcher software,a CpG island was found in the promoter region of theDAZ1 gene.The methylation status of this region was analyzed in sperm and leukocytes by bisulfited sequencing.Results:The methylation status of the CpG island in the DAZ1 gene promoter region differed in leukocytes andsperm:it was methylated in leukocytes,but unmethylated in sperm.Conclusion:A differentially methylated region ofthe DAZ1 gene exists in spermatic and somatic cells,suggesting that methylation of this region may regulate DAZ1gene expression in different tissues.(Asian J Androl 2006 Jan;8:61-67)     

Aberrant methylation of the TDMR of the GTF2AIL promoter does not affect fertilisation rates via TESE in patients with hypospermatogenesis

Increasing evidence shows a relationship between epigenetic regulation and male infertility. The GTF2A1L gene promoter contains the DNA methylation site of a tissue-specific differentially methylated region （TDMR）. Eighty-six patients with non-obstructive azoospermia were assessed for the DNA methylation state of CpG islands in the GTF2AIL promoter using testicular genomic DNA. Based on histological criteria, 26 of the 86 patients had normal spermatogenesis （controls）, 17 had hypospermatogenesis and 26 had a Sertoli cell-only phenotype or tubular sclerosis. GTF2AIL TDMR methylation was significantly lower in testes DNA from control samples than from hypospermatogenic samples （P=0.029）. Patients with hypospermatogenesis were divided into two subgroups： high DNA methylation （HM, n=5） and low DNA methylation （LM, n= 12）. The GTF2AIL TDMR methylation rate differed significantly between the HM and LM groups （P=0.0019）, and GTF2A 1L expression was significantly higher among the LM than in the HM patients （P=0.023）. High TDMR methylation was correlated with low GTF2AIL gene expression levels. Both groups demonstrated relatively good outcomes with respect to sperm retrieval, fertilisation, pregnancy and childbirth rates. We observed that aberrant GTF2AIL gene expression was not correlated with fertilisation rates. The testicular sperm extraction （TESE） technique may be used to overcome male infertility due to aberrant TDMR methvlation.     

上皮钙黏素1基因启动子甲基化对结肠癌上皮钙黏素和β-连接素表达的影响

目的 探讨上皮钙黏素基因(CDH1)启动子甲基化与结肠癌上皮钙黏素(E-cadherin)及β-连接素(β-catenin)的表达及临床病理特征的关系.方法 采用甲基化特异性PCR技术检测68例结肠腺癌组织、癌旁组织及正常黏膜组织中CDH1基因启动子甲基化的状况.采用免疫组织化学法检测E-cadherin及β-catenin蛋白的表达.结果 癌旁组织及癌组织中CDH1启动子甲基化的阳性表达分别为32.4%(22/68)、57.4%(39/68),正常组织均为阴性表达(P<0.05).E-cadherin在正常组织、癌旁组织及腺癌组织中阳性表达率分别为92.6%、66.2%和44.1%.正常组织中β-catenin均表达于细胞膜上,无胞质和(或)胞核表达,而β-catenin在癌旁组织及癌组织中胞质和(或)胞核表达分别为29.4%和50.0%.CDH1基因启动子甲基化阳性率与E-cadherin表达则呈负相关(r=-0.312,P=0.01),与β-catenin胞质和(或)胞核表达呈正相关(r=0.309,P=0.018).CDH1基因启动子甲基化及E-cadherin、β-catenin的异常表达均与结肠癌分化程度及转移密切相关(P<0.05).结论 CDH1基因启动子甲基化可能是导致结肠癌E-cadherin与β-catenin异常表达及肿瘤侵袭性增强的重要原因.

Abstract：

Objective To investigate the relationship between methylation of the CDH1 gene promoter on the expression of E-cadherin and β-catenin, and to evaluate the correlation with clinicopathological characteristics of the colonic carcinoma. Methods Methylation specific PCR (MSP) was used to detect CDH1 gene promoter methylation in the cancer tissue, adjacent tissues and normal tissues in 68 patients. The expression of E-cadherin and β-catenin was determined by immunohistochemistry staining. Results The positive rate of CDH1 gene promoter methylation was 32.4% in adjacent tissues and 57.4% in cancer tissue, while no detectable methylation was found in all the normal tissues. The difference was statistically significant. The positive rate of E-cadherin was 92.6% in the normal tissues, 66.2% in the adjacent tissues and 44.1% in the cancer tissues. In all normal tissues, β-catenin was expressed only at the cellular membrane but not in the cytosol or nucleus, while the expression of β-catenin was present in the cytosol or nucleus in 29.4% of the adjacent tissues and 50.0% of the cancer tissues. The positive rate of CDH1 gene promoter methylation was negatively correlated with E-cadherin expression (r =-0.312,P =0.01) and positively correlated with β-catenin cytosolic/nucleus expression(r=0.309,P=0.018). The differentiation and metastasis of colonic carcinoma were associated with the aberrant expression of E-cadherin, β-catenin, and methylation of CDH1 promoter (P<0.05). Conclusion CDH1 gene promoter methylation may lead to aberrant expression of E-cadherin and β-catenin in colonic carcinoma, and may play an important role in promoting the invasion of tumor.     

Demethylation of the γ-synuclein gene CpG island in colorectal cancer and its clinical significance

Objective To explore the relationship between γ-synuclein gene expression and CpG island demethylation in colorectal cancer (CRC), and the relationship between the demethylation and clinicopathological factors of CRC. Methods The expression of γ-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues (NNAT) by RT-PCR.CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-C). Before and after the treatment, the expression of γ-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of γ-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylationspecific PCR (real-time MSP). The relationship between the demethylation of γ-synuclein in CRC and clinicopathological factors was analyzed. Results The mean γ-synuclein mRNA expression was 0.66±0.34 in CRC samples, which was much higher than 0.45±0.26 in NNAT samples (P=0.011). 5-aza-C could induce expression and demethylation of γ-synuclein in COLO205, LoVo and SW480 cells. Γ-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples.The demethylated status of γ-synuclein was much higher in CRC samples than that in NNAT samples (P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC (P＜0.05). Conclusion The upregulation of γ-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.     

Demethylation of the γ-synuclein gene CpG island in colorectal cancer and its clinical significance

Objective To explore the relationship between γ-synuclein gene expression and CpG island demethylation in colorectal cancer (CRC), and the relationship between the demethylation and clinicopathological factors of CRC. Methods The expression of γ-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues (NNAT) by RT-PCR.CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-C). Before and after the treatment, the expression of γ-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of γ-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylationspecific PCR (real-time MSP). The relationship between the demethylation of γ-synuclein in CRC and clinicopathological factors was analyzed. Results The mean γ-synuclein mRNA expression was 0.66±0.34 in CRC samples, which was much higher than 0.45±0.26 in NNAT samples (P=0.011). 5-aza-C could induce expression and demethylation of γ-synuclein in COLO205, LoVo and SW480 cells. Γ-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples.The demethylated status of γ-synuclein was much higher in CRC samples than that in NNAT samples (P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC (P＜0.05). Conclusion The upregulation of γ-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.     

凋亡蛋白酶活化因子-1基因甲基化在骨肉瘤细胞株中的作用

目的 检测骨肉瘤细胞株中凋亡蛋白酶活化因子(APAF)-1基因启动子区域甲基化状态及该基因mRNA表达,观察APAF-1基因对骨肉瘤形成的影响.方法 以骨肉瘤细胞株MG63和Hs888T为研究对象,提取DNA,经重亚硫酸钠处理后,采用限制性酶切图谱分析(COBRA)检测APAF-1基因CpG岛的甲基化情况,使用不同浓度(0、1×10~(-7)、3×10~(-7)mol/L)的DNA甲基转移酶抑制剂5-氮杂2'-脱氧胞苷(5-Aza-CdR)处理骨肉瘤细胞株4、10、20d,采用实时定量聚合酶链反应(QT-PCR)检测Apaf-1 mRNA表达水平的变化.结果 在Hs888T细胞株中APAF-1基因存在CpG岛甲基化并且随着5-Aza-CdR浓度的增加Apaf-1 mRNA表达水平也在增加.3×10~(-7)mol/L的5-Aza-CdR处理Hs888T细胞株20 d与对照组比较差异有统计学意义(P<0.05).结论 Hs888T细胞株中APAF-1基因异常表达与APAF-1基因启动子区域CpG岛甲基化有关,提示APAF-1基因甲基化可能参与某些骨肉瘤的发生.     

TSA对人肝癌细胞株E-cadherin启动子区甲基化及表达的影响

目的 探讨去乙酰化转移酶抑制剂TSA对人肝癌SMMC-7721细胞株E-cadherin基因(E-cad)启动子区甲基化及其表达的影响.方法 TSA(300 nm/L)处理人肝癌SMMC-7721细胞,MTT法、TUNEL染色法分别检测细胞生长抑制及凋亡情况,甲基化特异性的PCR(methylation-specificPCR,MSP)检测处理前后E-cad启动子区CpG岛甲基化状态;Western blot检测处理前后E-cad及DNMT3b表达水平的变化.结果 TSA能抑制人肝癌SMMC-7721细胞生长并诱导其发生凋亡,与对照组相比,实验组细胞生长抑制率为21.85%,对照组细胞凋亡率为(4.69±0.56)%,实验组凋亡率为(14.94±0.91)%,与对照组相比,凋亡细胞明显增多(P=0.000).用药前E-cad启动子区CpG岛为甲基化状态,E-cad蛋白表达阴性.TSA处理后E-cad启动子区CpG岛发牛脱甲基化,E-cad蛋白恢复表达.TSA亦导致DNMT3b的表达水平降低. 结论去乙酰化转移酶抑制剂TSA能抑制人肝癌SMMC-7721细胞生长,并可诱导其发生凋亡,逆转E-cad基因启动子区的甲基化状态,并恢复该基因表达.TSA有可能通过DNMT3b发挥脱甲基化作用.     

原发性肝癌RUNX3基因启动子区甲基化及mRNA表达及其意义

目的 观察原发性肝癌中人RUNT相关转录因子3(RUNX3)基因启动子甲基化及mRNA表达,探讨其甲基化与临床特征的关系.方法 收集75例原发性肝癌患者的肿瘤标本及其癌旁组织、10例正常肝组织,应用甲基化特异性聚合酶链反应(MSP)测定RUNX3基因CpG岛甲基化状态,并采用实时定量聚合酶链反应(PCR)分析RUNX3基因在75例原发性肝癌中的表达水平及甲基化与肝癌临床特征的关系.结果 75例肝癌组织中有34例(45.3％)存在RUNX3基因CpG 岛的异常甲基化,癌旁组织中有7例(9.3％),而正常肝组织中未检测到RUNX3基因CpG岛的异常甲基化,RUNX3基因异常甲基化在肝癌组织、癌旁组织及正常组织中的发生率差异有统计学意义(x2=29.18,P＜0.01);75例原发性肝癌实时定量PCR分析有45例肝癌组织中存在RUNX3 mRNA 表达缺失或下调,其中60％ (27/45)伴随启动子甲基化,即RUNX3 mRNA表达下调与RUNX3基因甲基化密切相关(x2=9.77,P＜0.01);而肝癌组织非甲基化组RUNX3基因表达量高于甲基化组4倍以上;RUNX3基因CpG岛甲基化与患者肝硬化关系密切(x2=5.07,P＜0.05).结论 原发性肝癌存在RUNX3基因CpG岛异常甲基化,CpG岛的甲基化可能是导致其基因表达降低的主要原因之一,并与患者肝硬化密切相关.     

胰腺癌微卫星不稳定性、hMLH1启动子甲基化及其蛋白表达研究

目的探讨胰腺癌中微卫星不稳定性（MSI）与hMLH1启动子甲基化及蛋白表达之间的联系，揭示胰腺癌发生的分子机制。方法从35例胰腺癌患者的正常胰腺组织、癌组织中提取DNA；SSCP法检测标本中微卫星不稳定性发生情况；免疫组织化学法检测错配修复基因hMLH1在胰腺癌中的表达情况；MSP法检测hMLH1基因启动子甲基化状态。结果35例胰腺癌中微卫星高度不稳定7例，低度不稳定14例，稳定11例，正常组织中没有出现微卫星不稳定，两者之间差异有统计学意义（P〈0．05）。hMLH1在微卫星不稳定胰腺癌组织中常为缺失表达。在微卫星稳定胰腺癌组织中呈正常表达。35例胰腺癌中hMLH1启动子CpG岛甲基化发生率为60％（21/35）。正常组织中未发现甲基化，两者之间差异有统计学意义（P〈0．05）。结论与胰腺癌有关的错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制，而hMLH1的表达失活则可能导致MSI的产生，促进胰腺癌的发生。     

抑癌基因RUNX3在膀胱癌T24细胞株中的表达以及对凋亡的影响

目的 明确RUNX3抑癌基因在正常膀胱组织和膀胱癌T24细胞株的甲基化状态,并将野生型RUNX3基因转染T24细胞,探讨RUNX3基因恢复表达后对膀胱癌细胞凋亡的影响. 方法应用RT-PCR、甲基化特异性PCR和非甲基化PCR检测正常膀胱组织、膀胱癌T24细胞中RUNX3基因的表达以及基因启动子区域CpG岛的甲基化状态.构建真核载体的RUNX3-EGFP-pDest质粒,在LipefectamineTM2000介导下转染膀胱癌T24细胞,转染实验设立3组:空白对照组、空质粒EGFP-pDest转染组以及RUNX3-EGFP-pDest转染组.Western blot检测RUNX3蛋白表达,流式细胞仪检测细胞凋亡情况. 结果正常膀胱组织RT-PCR可检测出1248bp的RUNX3基因条带,而膀胱癌T24细胞中无法检测出RUNX3基因的表达.正常膀胱组织RUNX3基因甲基化PCR检测阴性,非甲基化PCR阳性;T24细胞反之.正常膀胱粘膜组和RUNX3-EGFP-pDest质粒转染组Western blot检测,均表达RUNX3蛋白,而膀胱癌T24细胞未表达RUNX3蛋白.流式细胞仪检测,空白对照组的凋亡率为(3.1±0.46)%,而EGFP-pDest转染组和RUNX3-EGFP-pDest质粒转染组的凋亡率分别为(10.1±1.62)%、(41.20±1.53)%,应用方差分析的LSD法和SNK法进行均数的多重两两比较,RUNX3-EGFP-pDest质粒转染组与EGFP-pDest转染组、空白对照组凋亡率之间的差异均具有显著性,P<0.01.结论 正常膀胱组织RUNX3基因正常表达,未发生启动子区域CpG岛甲基化,而膀胱癌T24细胞可能因RUNX3基因启动子区域CpG岛发生甲基化,致RUNX3基因无法表达.转染野生型RUNX3抑癌基因后促进了膀胱癌T24细胞的凋亡.     

ER基因CpG岛甲基化的表达及其临床意义

目的:研究ER基因表达及CpG岛甲基化状况与胃癌生物学行为和预后的关系.方法:分别应用免疫组化SP法和限制性内切酶PCR法分析91例胃癌ER的表达和30例胃癌ER墓因CpG岛甲基化的状况.结果:胃癌ER阳性率为38%(35/91),其中高分化腺癌10%(3/30),分别与低分化腺癌43%(13/30)、未分化腺癌53%(8/15)、粘液癌63%(5/8)及印戒细胞癌75%(6/8)比较皆具有显著性差异(P＜0.05);伴淋巴结转移组为55%(11/20),未转移组22.5%(9/40),差异有显著性(P＜0.05).正常胃粘膜ER基因CpG岛未甲基化,而胃癌呈高度甲基化40%(12/30)(P＜0.05).结论:ER在胃癌中的表达与浸润、转移有关,ER阳性胃癌生物学行为差,ER基因CpG岛甲基化可能是胃癌中ER失表达的分子机制.     

胰腺癌SOCS-1基因异常甲基化研究

目的：探索人类胰腺癌中SOCS—1基因是否由于异常甲基化而表达抑制；查询此现象在胰腺癌的发生、发展中的意义。方法：采集25例胰腺导管腺癌病人的肿瘤标本及5例对应的正常胰腺组织，运用甲基化特异性PCR反应研究胰腺癌组织中SOCS—1基因CpG岛甲基化状态，同时运用实时定量PCR分析SOCS—1基因的表达。结果：25例胰腺导管腺癌病人中有9例（36％）SOCS—1基因呈CpG岛甲基化，而正常组织则无SOCS—1基因CpG岛甲基化；SOCS—1基因CpG岛甲基化组的SOCS—1基因表达量与无SOCS—1基因CpG岛甲基化组相比，其基因相对表达量明显减少（P〈0．05），证明SOCS—1基因CpG岛甲基化可以抑制SOCS—1基因表达。与病人临床病理特征相结合比较．发现SOCS—1基因CpG岛甲基化与年龄、性别、肿瘤体积、肿瘤分化程度及TNM分期等因素无关。结论：在胰腺导管腺癌中存在SOCS—1基因CpG岛甲基化，且由于CpG岛甲基化而促使基因表达抑制。SOCS—1基因CDG岛甲基化在胰腺癌的发生、发展中可能具有一定的作用。     

5-杂氮-2′-脱氧胞苷对前列腺癌PC-3细胞生物学行为的影响

目的 检测E-cadherin基因在激素非依赖性前列腺癌(HIPC)细胞上的表达及启动子CpG岛甲基化,探讨甲基化抑制剂5-杂氮-2′-脱氧胞苷(5-Aza-CdR)对HIPC细胞的影响及意义.方法 2.5、5.0、10.0 μmol/L的5-Aza-CdR处理PC-3细胞72h后,甲基化特异性聚合酶链反应(MSP)方法检测CpG岛甲基化改变,逆转录—聚合酶链反应(RT-PCR)方法检测E-cadherin mRNA变化,Westernblot方法检测E-cadherin蛋白变化,Transwell小室检测细胞侵袭性改变.结果 HIPC的E-cadherin启动子CpG岛甲基化呈阳性,基因表达缺失,细胞侵袭性明显.经5-Aza-CdR作用之后,CpG岛甲基化阳性明显减弱(P＜0.05),E-cadherin基因恢复表达(P＜0.05),PC-3细胞的侵袭性下降约59.68％,且与药物的浓度呈正相关.结论 5-Aza-CdR可逆转PC-3细胞E-cadherin启动子CpG岛的异常甲基化,诱导mRNA转录和蛋白的表达,并降低癌细胞的侵袭性.