Reduced T-cadherin expression and promoter methylation are associated with the development and progression of hepatocellular carcinoma

Loss of T-cadherin expression has been reported in a number of human cancers. We previously reported that T-cadherin re-expression suppressed cell growth and motility in glioma. Here, we report that the T-cadherin expression was significantly decreased in human hepatocellular carcinoma (HCC) compared to adjacent normal liver. In addition, T-cadherin expression in HCC with metastasis was significantly lower than in HCC without metastasis. To determine the mechanism underlying the reduced T-cadherin expression in HCC, we examined T-cadherin promoter methylation. We found that methylation of the T-cadherin promoter was present in 40% of HCC, but absent in all adjacent liver tissues. In the HCC with T-cadherin promoter methylation, the T-cadherin expression was significantly decreased compared to HCC without methylation. To provide a functional link between T-cadherin promoter methylation and T-cadherin growth regulation, we used the HepG2 hepatoma cell line that exhibits T-cadherin promoter methylation. Treatment of HepG2 cells with the demethylating agent 5-aza-2-deoxycytidine resulted in increased T-cadherin expression and reduced cell proliferation. These results demonstrate that the T-cadherin down-regulation by promoter methylation is associated with the development and progression of HCC, and suggest that T-cadherin is an important tumor suppressor in liver cancer.     

Downregulation of expression of a novel cadherin molecule,T-cadherin,in basal cell carcinoma of the skin

T-cadherin appears to act as a tumor-suppressor factor in various cancers. Downregulation of T-cadherin is caused by a combination of allelic loss and hypermethylation of the T-cadherin promoter region and is related to cancer invasion. To elucidate the molecular mechanism of invasiveness of basal cell carcinoma of the skin, T-cadherin expression was investigated in archival pathological tissue sections made up of normal counterparts of skin and various types of basal cell carcinoma. Immunohistochemical staining showed that T-cadherin was not expressed in 38 of 51 (75%) basal cell carcinoma specimens, whereas normal counterparts of the skin appeared to express abundant T-cadherin. Loss of heterozygosity in intron 1 of the T-cadherin gene was found in four of 20 informative cases that did not express T-cadherin. Aberrant methylation of the T-cadherin promoter region also was found in six of 25 basal cell carcinomas by methylation-specific polymerase chain reaction. In contrast, no structural alternations were found in two loss of heterozygosity-positive basal cell carcinomas on sequence analysis. These findings indicated that T-cadherin expression was downregulated by a combination of allelic loss and aberrant methylation in basal cell carcinoma of the skin. Loss of T-cadherin expression might be related to the biological behavior of basal cell carcinoma. In addition, results of the present study suggested that downregulation of T-cadherin in various cancers might be related to tumor invasiveness rather than metastasis, because basal cell carcinoma of the skin principally lacks metastatic activity.     

结肠癌T-cadherin表达水平分析与5-Aza-CdR对其表达和结肠癌细胞增殖、侵袭及凋亡的影响

背景与目的：结肠癌是临床常见恶性肿瘤,探讨抑癌蛋白T-cadherin在结肠癌中的表达情况及其与患者临床病理学特征的关系,并分析5-Aza-CdR对T-cadherin表达和结肠癌细胞增殖、侵袭及凋亡的影响。方法：收集福建医科大学附属第二医院2015—2016年40例手术切除的结肠癌组织及癌旁组织新鲜样本,并经过病理学检查验证,分别提取总RNA和总蛋白质。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）分析T-cadherin mRNA的表达,采用蛋白质印迹法（Western blot）分析T-cadherin蛋白水平,并分析T-cadherin的mRNA表达变化与患者临床病理学特征的关系。进一步以人结肠癌细胞系HT-29为研究对象,采用甲基化抑制剂5-Aza-CdR处理HT-29细胞,分别采用RTFQ-PCR和Western blot分析T-cadherin表达变化,采用细胞计数试剂盒（cell counting kit-8,CCK-8）分析细胞增殖,采用Transwell实验验证细胞侵袭能力,采用流式细胞术分析细胞凋亡。结果：T-cadherin在结肠癌组织的蛋白水平和mRNA表达均明显低于癌旁组织,其mRNA表达与淋巴结转移（F=5.316,P=0.009 3）和分化程度（F=5.807,P=0.006 4）明显相关,而与其他病理变量（包括性别、年龄、肿瘤大小和肿瘤浸润深度）无明显相关。药物5-Aza-CdR可以显著上调HT-29细胞中T-cadherin的表达水平,抑制HT-29细胞增殖、侵袭并促进细胞凋亡。结论：T-cadherin表达可能与结肠癌恶性程度密切相关,药物5-Aza-CdR处理可上调结肠癌细胞T-cadherin的表达,并抑制结肠癌细胞的增殖、迁移,促进结肠癌细胞凋亡,提示T-cadherin可能是结肠癌患者使用甲基化抑制剂5-Aza-CdR治疗的靶点。     

siRNA敲减DNMT1表达对T-cadherin基因甲基化及结肠癌细胞生物学行为的影响

目的 肿瘤的发生、发展和侵袭转移是影响结肠癌治疗的重要原因,有研究发现DNA甲基化状态在肿瘤的发生、发展中起着重要作用,抑制T-cadherin表达可增强肿瘤细胞侵袭和迁移能力.本研究探讨siRNA敲减DNA甲基转移酶1(DNA methyl transferase 1,DNMT1)基因表达对结肠癌细胞T-cadherin基因甲基化及细胞生物学行为影响.方法 设计针对DNMT1的siRNA1、siRNA2和siRNA3重组质粒,分别转染HT-29、SW620和HCT116细胞系,采用实时荧光定量PCR检测DNMT1表达,以筛选敲减效率最高的DNMT1 siRNA和细胞系.用敲减效率最高的DNMT1 siRNA转染相应细胞系,采用甲基化特异性PCR检测T-cadherin甲基化水平,采用实时荧光定量PCR和蛋白印迹法检测T-cadherin表达.用敲减效率最高的DNMT1 siRNA转染相应细胞系,采用噻唑蓝比色法、流式细胞术、Transwell和细胞划痕实验检测细胞增殖、凋亡、侵袭和迁移情况,观察敲减DNMT1对细胞生物学行为的影响;同时设置T-cadherin shRNA重组质粒转染组,观察同时敲减DNMT1和T-cadherin对细胞生物学行为的影响.结果 DNMT1 siRNA1重组质粒对HT-29细胞系DNMT1的敲减效果最好.DNMT1 siRNA1转染HT-29细胞后,DNMT1组T-cadherin基因甲基化水平明显低于空质粒组和空白组,DNMT1组与空质粒组(F=314.182)和空白组(F=283.625)差异有统计学意义,均P<0.001.DNMT1组和联合组DNMT1 mRNA表达量(0.27±0.08)和蛋白表达量(0.41±0.12)显著低于空质粒组和空白组.其中mRNA表达,DNMT1组与空质粒组(F=544.581)和空白组(F=525.811)组差异有统计学意义,均P<0.001;联合组与空质粒组(F=507.501)和空白组(F=494.958)也差异有统计学意义,均P<0.001.蛋白表达量,DNMT1组与空质粒组(F=217.550)和空白组(F=275.469)差异有统计学意义,均P<0.001:联合组与空质粒组(F=321.707)和空白组(F=407.225)也差异有统计学意义,均P<0.001:而DNMT1组和联合组比较mRNA(F=0.002,P=0.965)和蛋白表达量(F=0.811,P=0.394)均差异无统计学意义.DNMT1组T-cadherin mRNA表达量(1.17±0.34)和蛋白表达量(1.38±0.54)显著高于联合组、空质粒组和空白组.其中mRNA表达,DNMT1组与联合组(F=433.103)、空质粒组(F=313.768)、空白组(F=372.118)差异有统计学意义,均P<0.001.对于蛋白表达量,DN-MT1组与联合组(F=89.315)、空质粒组(F=156.745)、空白组(F=196.702)差异有统计学意义,均P<0.001.而联合组与空质粒组(F=1.310,P=0.262;F=0.144,P=0.714)和空白组(F=3.713,P=0.064;F=0.038,P=0.850)的mRNA和蛋白表达量比较差异无统计学意义.与联合组、空质粒组和空白组比较,DNMT1组第24、36、48、72 h的A值显著减小,F组别=14.479,P<0.001;F时间=37.755,P<0.001.DNMT1组与联合组(F=1004.194,P<0.001)、空质粒组(F=1190.815,P<0.001)、空白组(F=781.018,P<0.001)比较细胞总凋亡率显著升高;DNMT1组穿膜细胞数目为(61.3±10.7)个,与联合组(F=86.730,P<0.001)、空质粒组(F=282.962,P<0.001)、空白组(F=152.538,P<0.001)比较显著减少.DNMT1组细胞迁移距离为(235.8±6.3)μm,与联合组(F=2535.55,P<0.001)、空质粒组(F=9767.233,P<0.001)、空白组(F=8420.830,P<0.001)比较显著减少.而联合组与空质粒组和空白组比较,A值、细胞总凋亡率、穿膜细胞数目和细胞迁移距离均差异无统计学意义,P>0.05.结论 DNMT1 siRNA1可有效敲减HT-29细胞DNMT1表达,逆转T-cadherin高甲基化状态并上调其表达.敲减DNMT1可明显抑制HT-29细胞增殖,促进其凋亡,降低其侵袭和迁移能力;而同时敲减T-cadherin能够逆转此种现象,说明敲减DNMT1引起的细胞生物学行为改变可能是通过T-cadherin来实现的.     

Klotho,an anti-senescence related gene,is frequently inactivated through promoter hypermethylation in colorectal cancer

The potential anti-senescence gene Klotho (KL) has been recently found to participate in the progression of several different human cancers including breast, lung, and cervical cancer. In this current study, we identified KL as a candidate tumor suppressor gene silenced through promoter hypermethylation in colorectal cancer (CRC). KL gene expression is found to be absent or reduced in colon cancer cell lines (5/6, 83.3%), which can be reversed by treatment with demethylation agent 5-aza-2′-deoxycytidine (Aza), but not HDAC inhibitor trichostatin A. In addition, KL expression is markedly downregulated in colorectal carcinoma tissues when compared to the adjacent nontumor tissues (n = 25, p < 0.001). The methylation of the KL gene promoter was frequently detected in primary tumor tissues (34/40, 85%) when compared with adjacent nontumor colon tissues. Furthermore, ectopic expression of KL led to the cell proliferation inhibition of colon cancer cell lines via the induction of cell apoptosis and S-phase cell cycle arrest. Taken together, our results suggest that KL is inactivated through promoter hypermethylation and potentially functions as a tumor suppressor gene in CRC.     

The involvement of CHD5 hypermethylation in laryngeal squamous cell carcinoma

Chromodomain helicase DNA-binding protein 5 (CHD5) has been found to be a candidate tumor suppressor gene (TSG) in malignant neural tumors. In mice heterozygous for chd5 deficiency, the first tumor observed was pathological squamous cell carcinoma. More than 95% of primary laryngeal cancer is squamous cell carcinoma. Thus, we explored the expression of CHD5 in 65 patients with laryngeal squamous cell carcinoma (LSCC) using real-time PCR, immunohistochemistry and Western blotting. DNA methylation was detected using bisulfate-specific sequencing. The potential function of CHD5 was determined using MTT, apoptosis and transwell migration assays in CHD5-transfected Hep-2 cells. Our results revealed that the mRNA and protein expression levels of CHD5 in LSCC tissues were significantly lower than those in clear surgical margin tissues (p<0.05), and there is a significant correlation between the mRNA and protein expression levels of CHD5 (p<0.01). In addition, there were significant differences in CHD5 mRNA and protein levels with respect to the patient's clinical stage (p<0.05). Aberrant methylation of the CHD5 promoter was frequently found in the Hep-2 cell line and LSCC tumor tissues, especially tumor tissues from advanced TNM (p<0.05) or older patients (p<0.05). Finally, ectopic expression of CHD5 in laryngeal cancer cells led to significant inhibition of growth and invasiveness. Our data suggest that CHD5 is a tumor suppressor gene that is epigenetically downregulated in LSCC.     

Silencing of the UCHL1 gene in human colorectal and ovarian cancers

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2'-deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (> or =8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.     

UGCG通过调控MDR1/P-gp参与人结肠癌奥沙利铂耐药的初步研究

目的 建立人结肠癌耐奥沙利铂（L-OHP）细胞株,检测该细胞株的多药耐药性并初步探讨其可能的耐药机制。方法 以人结肠癌细胞HCT116为对象,采用药物浓度梯度递增诱导法建立人结肠癌耐奥沙利铂细胞株HCT-116/L-OHP。CCK-8法检测L-OHP、顺铂（DDP）、5-氟尿嘧啶（5-Fu）对亲本细胞和耐药细胞株的半数抑制浓度（IC50）。使用UDP-葡萄糖神经酰胺糖基转移酶（UGCG）siRNA转染HCT-116/L-OHP细胞,实时荧光定量 PCR和Western blot检测干扰前后UGCG基因和多药耐药基因1（MDR1）mRNA及其编码的蛋白表达水平。结果 HCT-116/L-OHP对L-OHP的耐药指数为10.5,与DDP有一定程度的交叉耐药,耐药指数为4.61,但对5-Fu无交叉耐药。耐药细胞HCT-116/L-OHP中UGCG、MDR1 mRNA和UGCG、P-糖蛋白（P-gp, MDR1编码的蛋白）表达均增加,相比HCT-116细胞,差异具有统计学意义（P<0.05）。UGCG siRNA成功抑制HCT-116/L-OHP细胞中UGCG的表达,各干扰组MDR1 mRNA、P-gp表达减少,与对照组相比差异具有统计学意义（P<0.05）。结论 成功构建了人结肠癌耐药细胞株;UGCG基因通过调控MDR1/P-gp的表达参与人结肠癌奥沙利铂的耐药机制的形成。     

Frequent inactivation of SPARC by promoter hypermethylation in colon cancers

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.     

鼻咽癌细胞株14-3-3σ启动子去甲基化与基因表达研究

目的 研究鼻咽癌细胞株14-3-3σ基因启动子甲基化状况及去甲基化处理对其表达的影响.方法 用甲基化特异性聚合酶链反应(PCR)和逆转录PCR方法检测鼻咽癌细胞株CNEI、CNE2、5-8F、6-10B和非肿瘤人鼻咽上皮细胞NP69细胞14-3-3σ基因启动子甲基化状况和mRNA表达水平.用不同浓度5-杂氮-2'-脱氧胞苷(5-aza-2dC)处理鼻咽癌细胞株72 h,检测处理组和对照组14-3-3σ基因启动子甲基化状况和mRNA表达水平,并用Western-blot检测14-3-3σ蛋白表达情况.结果 NP69细胞14-3-3σ启动子未检测到甲基化,CNE1、CNE2、5-8F、6-10B细胞14-3-3σ基因启动子都存在甲基化,4株肿瘤细胞的14-3-3σ mRNA表达水平显著低于NP69细胞.5-aza-2dC处理能剂量依赖性地逆转鼻咽癌细胞14-3-3σ启动子的甲基化状态并上调其mRNA和蛋白质的表达水平,高分化细胞株(CNE1)对药物的敏感性高于低分化细胞株(CNE2、5-8F和6-10B).结论 启动子甲基化是导致鼻咽癌细胞株14-3-3σmRNA和蛋白质表达降低的直接原因,14-3-3σ启动子去甲基化可能成为鼻咽癌治疗的新靶点.     

人乳腺癌细胞系RUNX3基因启动子甲基化状态研究

目的：探讨人乳腺癌细胞系中抑癌基因RUNX3启动子甲基化状态,并分析其与RUNX3基因表达的相关性。方法：运用甲基化特异性PCR（MSP）检测5种乳腺癌细胞系和一种人类正常乳腺细胞系中RUNX3启动子甲基化状态,运用RT-PCR和Western印迹检测这些细胞系中RUNX3基因mRNA和蛋白的表达。结果：在6种细胞系中,有两种（T47D、MCF7）呈高甲基化状态,并且这两种细胞系的RUNX3 mRNA和蛋白表达阴性。SKBR3中没有检测出RUNX3启动子区的甲基化,但RUNX3 mRNA和蛋白表达阴性。结论：乳腺癌细胞系中RUNX3基因由于启动子区甲基化而失活,但尚有其它失活机制存在,需进一步研究探讨。     

Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.     

DR4基因启动子甲基化对TRAIL诱导肺鳞癌细胞凋亡作用的影响

目的:探讨肺鳞癌中死亡受体4（DR4）不同甲基化状态是否会影响肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肺鳞癌细胞的诱导凋亡作用。方法:采用甲基化特异性PCR（MSP）、RT-PCR和Western blot法检测5-氮-2′-脱氧胞苷（5-Aza-CdR）处理肺鳞癌细胞株（H226、SK-MES-1、H520）前后DR4基因启动子甲基化状态和基因表达情况;MTT法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡率。结果:MSP、RT-PCR和Western blot结果表明H226、SK-MES-1细胞DR4基因呈甲基化状态,其mRNA及蛋白均低表达,H520细胞中DR4基因呈非甲基化状态,其mRNA及蛋白高表达;5-Aza-CdR干预后H226、SK-MES-1细胞DR4基因呈非甲基化状态,其mRNA及蛋白表达较前均显著上调,差异有统计学意义（P＜0.05）,H520仍呈非甲基化状态,其mRNA及蛋白表达量较前无明显差异。同时研究还证实TRAIL对H226、SK-MES-1细胞有不同程度的细胞增殖抑制率和凋亡率,但不敏感,5-Aza-CdR干预后,H226及SK-MES-1细胞对TRAIL的敏感性较前均显著增高（P＜0.05）。结论:5-Aza-CdR可以逆转肺鳞癌中DR4基因启动子甲基化状态,进而增加TRAIL诱导肺鳞癌细胞凋亡的作用。5-Aza-CdR联合TRAIL可能是治疗肺鳞癌的一种新策略。     

β-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells

Purpose

Over half of the colon cancer patients suffer from cancer-related events, mainly metastasis. Loss of β-catenin activity has previously been found to facilitate cancer cell dissociation and migration. Here, we aimed to investigate whether epigenetic silencing of β-catenin induces human colon cancer cell migration and/or invasion.

Methods

HCT-116, Caco-2, HT-29 and SW620 cell migration and invasion capacities were assessed using scratch wound healing and Matrigel invasion assays, respectively. Confocal microscopy, qRT-PCR and Western blotting were performed to determine gene expression levels, whereas methylation-specific quantitative real-time PCR was used to assess the extent of β-catenin gene (CTNNB1) promoter methylation after treatment of the cells with TPA, hydrogen peroxide, 5-aza-2′-deoxycytidine and/or VAS2870.

Results

We found that treatment of HT-29 and Caco-2 cells (differentiated and low metastatic) with 12-O-tetradecanoyl phorbol-13-acetate (TPA; a tumor promoter) suppressed E-cadherin and β-catenin expression at both the mRNA and protein levels and, in addition, enhanced cell migration. Furthermore, we found that the CTNNB1 gene promoter methylation levels were higher in the more invasive HCT-116 and SW620 colon cancer cells than in HT-29 and CCD-841 (normal colon epithelial) cells. We also found that TPA or hydrogen peroxide induced CTNNB1 gene promoter methylation to a higher extent in HT-29 and CCD-841 cells than in HCT-116 and SW620 cells, and that the degree of CTNNB1 gene promoter methylation positively correlated with cell dissociation and migration. In addition, we found that co-treatment with 5-aza-2′-deoxycytidine (decitabine, a DNA methyl transferase inhibitor) and VAS2870 (a NADPH oxidase inhibitor) almost completely blocked the invasion of TPA-treated HT-29 and TPA-untreated HCT-116 and SW620 cells, and that these inhibitions surpassed those of the cells treated with decitabine or VAS2870 alone.

Conclusions

From our data we conclude that the extent of CTNNB1 gene promoter methylation by reactive oxygen species correlates with the migratory and invasive abilities of colon cancer cells. Our results suggest that epigenetic regulation of CTNNB1 may serve as a novel avenue to block colon cancer cell migration and invasion.

     

DNA 甲基化抑制剂对肿瘤细胞中核糖核酸酶抑制因子表达下调的影响

背景与目的:人核糖核酸酶抑制因子 (ribonucleasE-inhibitor, RI) RI能有效抑制血管生成因子诱导的血管形成及某些可移植性实体瘤在动物体内的生长.然而, RI抗肿瘤的分子机制还未完全阐明.许多抑癌基因通过启动子区域异常的甲基化而使表达缺失,去甲基化抑制能使其表达恢复.为了进一步了解 RI的功能以及探讨 RI与肿瘤发生的关系,本实验拟研究甲基化抑制剂 5-aza-2′-deoxycytidinE-(5-Aza-CdR)对肿瘤细胞中 RI表达的影响.方法:用 5-Aza-CdR作用人乳腺癌细胞系 MCF-7、人胃癌细胞系 BGC-823、人的前列腺癌细胞系 DU-145和人结肠癌细胞系 HT-29.通过 RT PCR,蛋白免疫印迹法( Western blot) ,免疫荧光( immunofluorescence)和免疫细胞化学( immunocytochemistry)技术分析 RI基因的表达.结果:与对照组比较, 5-Aza-CdR能显著提高 RI基因在 MCF-7、 BGC-823和 DU 145细胞中的表达, RT PCR检测结果分别为 37.2%、 46.0%和 32.4%, Western blot检测结果分别为 26.4%、 20.9%和 24.4%( P< 0.01);但对 HT-29细胞没有明显的影响.结论: RI基因可能与胃癌、前列腺癌和乳腺癌的发生有关.     

Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily.

The retinoblastoma protein-interacting zinc finger gene RIZ1 is a tumor suppressor gene and a member of a nuclear histone/protein methyltransferase superfamily. RIZ1 inactivation is commonly found in many types of human cancers and occurs through loss of mRNA expression, frameshift mutation, chromosomal deletion, and missense mutation. RIZ1 is also a tumor susceptibility gene in mice. We now show that loss of RIZ1 mRNA in human cancers is associated with DNA methylation of its promoter CpG island. Methylation of the RIZ1 promoter strongly correlated with lost or decreased RIZ1 mRNA expression in breast, liver, colon, and lung cancer cell lines as well as in liver cancer tissues. Treatment with the methylation inhibitor 5-aza-2'-deoxycytidine activated RIZ1 mRNA expression in cancer cells. Furthermore, methylation was found in 11 of 25 (44%) breast cancer specimens and 20 of 32 (62%) liver cancer specimens. Our results suggest that DNA methylation is a common mechanism in inactivating the RIZ1 tumor suppressor gene in human liver and breast cancers.     

乳腺癌中NDRG-1基因甲基化及其体外逆转研究

目的:探讨N-myc下游调节基因(N-myc downstream regulated gene-1,NDRG-1)基因在乳腺癌组织中的甲基化状态,研究甲基化酶抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-2'-deoxycytidine, 5-Aza-CdR)对乳腺癌细胞株T47D的生长增殖及NDRG-1 mRNA表达的影响.方法:采用甲基化特异性PCR(methylation specific PCR,MSP)法检测乳腺癌组织、相应癌旁组织和乳腺良性病变组织中NDRG-1基因启动子甲基化状态;5-Aza-CdR处理T47D细胞后,MTT法观察细胞生长活性的变化,RT-PCR法检测处理前后抑癌基因NDRG-1的mRNA表达变化.结果:NDRG-1基因在乳腺癌组织中甲基化率为46.8%,癌旁组织中甲基化率为21.3%,而乳腺良性病变组织均未检测到甲基化.T47D细胞经5-Aza-CdR处理后,与对照组相比,细胞生长受到明显抑制.RT-PCR检测发现,与对照组相比,不同浓度处理组细胞的NDRG-1 mRNA表达增多. 结论:NDRG-1基因甲基化状态与乳腺癌发生有密切关系.5-Aza-CdR逆转T47D细胞NDRG-1基因甲基化,恢复该基因的表达,从而抑制肿瘤细胞生长.