Combined analysis of E-cadherin gene (CDH1) promoter hypermethylation and E-cadherin protein expression in patients with gastric cancer: implications for treatment with demethylating drugs.

BACKGROUND: Hypermethylation is studied as a new, relevant mechanism for silencing tumor suppressor genes. It is a potentially reversible epigenetic change and it is the target of novel anticancer compounds with demethylating activity. In this perspective, we investigated E-cadherin gene (CDH1) promoter hypermethylation in gastric carcinomas and its correlation with E-cadherin protein expression. METHODS: Consecutive cases of gastric carcinoma with assessable paraffin-embedded tumor blocks and paired normal mucosa were considered eligible for study entry. CDH1 promoter hypermethylation and E-cadherin protein expression were determined by methylation-specific polymerase chain reaction and immunohistochemistry, respectively. RESULTS: CDH1 promoter hypermethylation was found in 20 out of 70 gastric carcinomas and the epigenetic change occurred in the early, as well as in the locally advanced disease. In five cases, hypermethylation was also detected in the normal mucosa. Eighteen out of 20 hypermethylated tumors were of the diffuse histotype (P=0.0001). Of 24 tumors with reduced or negative E-cadherin expression, 19 were hypermethylated and 5 were unmethylated (P=0.0001). CONCLUSIONS: CDH1 promoter hypermethylation frequently occurs in gastric carcinomas of the diffuse histotype and it is significantly associated with downregulated E-cadherin expression. The knowledge on the hypermethylation status of tumor suppressor genes may be relevant to the development of demethylating drugs and novel chemopreventive strategies in solid tumors.     

血液系统疾病中LRP15基因启动子区甲基化状态的研究

目的:探讨LRP15基因启动子区及第一外显子区的甲基化与血液系统疾病的关系和临床意义。方法:采用甲基化特异性PCR方法检测了9例正常人、73例急性白血病患者、8例慢性髓系白血病患者、2例慢性淋巴细胞白血病、12例血液系统良性病患者骨髓LRP15基因启动子区及第一外显子区的甲基化状况;硫化修饰结合限制性内切酶分析及硫化测序验证了一例血液系统良性病患者LRP15基因启动子区及第一外显子区的甲基化。结果:AL患者LRP15基因甲基化阳性率(71.23%)高于正常供者及慢性白血病(10%),差异有统计学意义(P<0.05)。LRP15基因在非恶性血液病患者中甲基化阳性率(55.56%)高于正常供者,硫化修饰结合限制性内切酶分析及硫化测序进一步证实了上述结论。结论:LRP15基因启动子的甲基化与不成熟的白血病细胞有关,与血液系统良性疾病也有一定关系。     

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。     

改进的亚硫酸氢钠测序法检测HepG2 RASSF1A基因CpG岛甲基化状态

目的 改进亚硫酸氢钠测序法，并检验改进后方法在甲基化检测中的可行性。方法 提取人肝癌细胞株HepG2基因组DNA，亚硫酸氢钠化学修饰，针对修饰后Ras相关家族1A基因（RASSF1A）启动子序列设计特异引物并结合沉降PCR扩增，T-A载体克隆、测序。结果 HepG2细胞RASSF1A基因启动子CpG岛的16个CpG位点均被甲基化。结论 改进后亚硫酸氢钠测序法能明显减少非特异性扩增，提高PCR效率，更适于基因甲基化状态的检测。     

RAS相关家族1A基因在胃癌中的表达和启动子甲基化

目的分析散发性胃癌中RAS相关家族1A基因(RASSF1A基因)的表达、突变和启动子甲基化状况,探讨RASSF1A基因在胃癌发生、发展中的意义.方法采用定量PCR、RT-PCR和SSCP的检测90例胃癌组织和30例相应癌旁正常组织中RASSF1A基因表达水平以及基因突变的情况;采用甲基化特异性PCR (MSP)方法检测RASSF1A基因启动子甲基化情况.结果 90例胃癌中有52例(57.8%)RASSF1A无表达或表达低下.RASSF1A无表达或表达低下和肿瘤细胞的分化(P<0.05)以及分期(P<0.001)相关,但是和肿瘤的浸润深度以及淋巴结转移不相关(P>0.05).90例胃癌中52例启动子发生甲基化(57.8%),其中90.3%(47/52)的RASSF1A无表达或表达低下组织中检测到RASSF1A基因启动子的甲基化,然而癌旁正常组织未发现有RASSF1A基因启动子的甲基化,SSCP没有发现任何突变.结论胃癌中存在较多的启动子异常甲基化造成RASSF1A基因失活,这可能是胃癌发生发展的因素之一.     

冷冻保存时长对卵裂期胚胎组蛋白表观遗传修饰的影响

【目的】研究不同的冷冻保存时长对人类卵裂期胚胎的组蛋白表观遗传修饰的影响。【方法】采用经患者知情同意捐献用于科研的慢速冷冻的卵裂期胚胎,根据冷冻时长分为6、9和12年组,以新鲜胚胎为对照组,每组6枚捐献胚胎。采用细胞免疫荧光技术观察去乙酰化酶HDAC1、组蛋白（H3K9ac、H3K4me3、H3K9me3）的表达量在各组胚胎中的差异,并以单细胞RT-qPCR技术检测去乙酰化酶HDAC1、甲基化酶（SUV39H1、SET?DB1）、去甲基化酶KDM5A对应mRNA的表达量。【结果】4组胚胎的去乙酰化酶HDAC1、组蛋白（H3K9ac、H3K4me3和H3K9me3）的蛋白表达量无统计学意义的差异（P>0.05）。同时去乙酰化酶HDAC1、甲基化酶SUV39H1对应mRNA的表达量也无统计学意义的差异（P>0.05）。随着胚胎冷冻时长的增加,甲基化酶SETDB1对应mRNA的表达量有递增趋势,然而差异无统计学意义（P>0.05）。去甲基化酶KDM5A对应的mRNA的表达量也随冷冻时长的增加而增强,差异有统计学意义（P<0.05）。【结论】慢速冷冻保存时长对卵裂期胚胎的组蛋白（H3K9ac、H3K4me3和H3K9me3）的蛋白表达量无显著影响,甲基化酶（SUV39H1、SETDB1）对应的mRNA表达量没有明显影响;但随着冷冻保存时长增加,胚胎去甲基化酶KDM5A对应的mRNA表达量增加,有可能导致胚胎转录抑制程度增强。     

5-aza-2’-deoxycytidine in the regulation of antioxidant enzymes in retinal endothelial cells and rat diabetic retina

AIM: To investigate the roles of a DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) in the regulation of antioxidant enzymes in diabetic retinopathy (DR) models. METHODS: DNMTs expressions and activity, and changes of two key antioxidant enzymes in DR, MnSOD (encoded by SOD2 gene) and glutathione S-transferase theta 1 (GSTT1), were quantified in the isolated human retinal endothelial cells (HRECs) exposed to high glucose (HG) with or without 5-aza-dC treatment. The downstream exacerbating factors including vascular endothelial growth factor (VEGF), intercellular adhesion molecule 1 (ICAM-1) and matrix metalloproteinase 2 (MMP2), which are implicated in the pathogenesis of DR and closely related to oxidative stress were also analyzed. The key parameters were confirmed in the retina from streptozotocin (STZ) diabetic rats. RESULTS: DNMTs expression and DNMT activity was induced in HRECs exposed to HG. Hyperglycemia decreased MnSOD and GSTT1 expression. 5-aza-dC administration effectively suppressed DNMTs expression and activity and reversed the MnSOD and GSTT1 expression under HG condition. VEGF, ICAM-1 and MMP2 induced by HG were also suppressed by 5-aza-dC treatment. Similar results were observed in the retina from STZ diabetic rats. CONCLUSION: Our findings suggest that DNA methylation may serves as one of the mechanisms of antioxidant defense system disruption in DR progression. Modulation of DNA methylation using pharmaceutic means such as DNMT inhibitors could help maintain redox homeostasis and prevent further progression of DR.     

Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP− primary glioblastoma

To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP− primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ?18 months) and 20 short-term survivors (STS; overall survival ?9 months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP− samples. Methylation levels of 11 CpG loci (10 genes) were statistically significantly lower, and 43 CpG loci (40 genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP− samples (P < 0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP− samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP− primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP− primary GBMs.