食管癌GATA基因家族高甲基化研究

背景 GATA转录因子家族在胚胎发育时期能够促进胃肠道的正常发育，并有助于在成年肠上皮的不断更新中调节组织分化，最近有报道在人胃肠肿瘤中GATA基因，即GATA—4，GATA—5的表达缺失。目的 探讨在食管肿瘤中渐进性的转录沉默而导致GATA相关基因表达缺失的情况。方法 用RT—PCR法检测GATA—4，—5，—6基因在17个食管鳞癌细胞系中的表达，从每个细胞系及88例原发性食管肿瘤标本中提取DNA，进行甲基化特异性PCR(methylation—specific PCR，MSP)，以检测GATA相关基因增强子区的甲基化情况，用5—aza—2’—deoxycytidine c5—aza—dc)在去甲基化的细胞系中进行检测GATA—4，—5基因的表达与增强子甲基化之间的关系，并对GATA增强子区进行亚硫酸氢盐测序以证实MSP的结果。结果 GATA—4／—5的表达在大多数食管癌细胞系中缺失，在每个未检测到GATA基因表达的细胞系中均可发现转录起始下游包括CpG岛在内的异常甲基化，GATA—6在每一细胞系中均表达。在正常食管粘膜中未发现GATA—4／—5增强子的甲基化，而GATA—4／—5的甲基化却可以在原发性食管癌中检测到。在27／44(61％)的食管鳞癌和31／44(71％)的食管腺癌中，发现GATA—4的甲基化，与此类似，GATA—5的甲基化也在一部分食管肿瘤中被检测到。结论 本文是关于继发于基因增强子高甲基化的食管肿瘤中，GATA—4，—5而不包括—6表达沉默的首篇报告。     

Silencing of EphA2 inhibits invasion of human gastric cancer SGC-7901 cells in vitro and in vivo

Receptor tyrosine kinases (RTKs), the common products of transforming oncogenes, have been widely used as indicators in the genesis and progression of human tumors. Until now, the erythropoietin-producing human hepatocellular (Eph) receptors have been recognized as the largest family of RTKs. EphA2, one member of Eph receptors, locates on human chromosome 1p36.1 which is a hot region for cancer research. It has been reported that high EphA2 expression levels were correlated with the tumor metastasis and poor prognosis. Increased expression of EphA2 can promote tumor growth and enhance the metastatic potential. To further define the function of EphA2 in malignant invasion, we employed the small interference RNA (siRNA) technique to knockdown gene expression of EphA2 in the gastric cancer SGC-7901 cell. Our results showed that the expression of double stranded RNA led to the efficient and specific inhibition of endogenous EphA2 expression in SGC-7901 cells. Silencing of EphA2 expression inhibited cell proliferation, caused cell cycle arrest, and decreased cell invasion in vitro. In addition, intratumoral injection EphA2 siRNA plasmid suppressed the growth of SGC-7901 cells xenografts in nude mice. Furthermore, knockdown of EphA2 expression reduced the expression of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo. In conclusion, our findings demonstrate that silencing of EphA2 inhibits gastric cancer SGC-7901 cell proliferation, invasion and MMP-9 expression, which indicate that the specific inhibition of EphA2 may be a potential approach for gastric cancer therapy.     

Research on the reactivation of Syk expression caused by the inhibition of DNA promoter methylation in the lung cancer

The aim of this study was to study the expression of Syk gene and methylation in its promoter region in the lung cancer and to investigate the relationship between silencing of the Syk gene and DNA methylation of the Syk promoter region in lung cancer cell lines. Real-time PCR and immunohistochemistry were used to examine the Syk expression in specimens from 3 lung cancer cell lines and 16 lung cancer patients (tumor tissues and adjacent normal tissues). MSP was used to analyze the methylation status of the Syk promoter region. We also investigated the role of restoring Syk expression by using a DNA methyltransferase inhibitor, 5-aza-CdR, in suppressing invasion of lung cancer cell lines. No expression of the Syk gene was detected in the 3 lung cancer cell lines. In the 16 lung cancer patient samples, Syk expression was significantly lower in the tumor tissues than that in their adjacent normal tissues (P<0.05). Consistently, immunohistochemistry analysis of Syk protein expression showed that in the lung cancer tissues Syk protein expression was also significantly lower than that in their adjacent normal tissues. In the two lung cancer cell lines (NL9980, YTMLC-9) that lack the endogenous Syk expression, 4uM demethylation agent 5-aza-CdR treatment was able to reactivate the Syk gene expression, but not NCI-H446. In conclusion, hypermethylation leads to silencing of the Syk gene in human lung carcinoma cell lines. Methylation of the Syk promoter and loss of Syk expression in lung cancer cell lines are independent biomarkers. Syk may be a potential tumor suppressor in human lung cancer.     

肝癌细胞中OY-TES-1基因启动子的甲基化状态

目的:研究肝癌细胞株OY-TES-1启动子的甲基化状态及甲基转移酶抑制剂对其启动子甲基化的影响.方法:运用生物信息学在线软件预测OY-TES-1启动子区域及转录因子结合位点;应用亚硫酸氢盐测序法(bisulfite-sequencing PCR,BSP)分析目的基因CpG位点的甲基化状态;用甲基转移酶抑制剂5-Aza-...     

TIP30基因启动子甲基化与大肠癌细胞5-氟尿嘧啶化疗敏感性的关系

目的探讨TIP30启动子CpG岛甲基化状态与大肠癌细胞对5-氟尿嘧啶(5-Fu)敏感性的关系。方法采用MTT法检测HCT116和HT29大肠癌细胞株对5-Fu的敏感性。应用甲基化特异性PCR(MSP)方法,检测2细胞株对5-Fu敏感性有差异的大肠癌细胞株中TIP30基因启动子CpG岛甲基化状态,并用RT-PCR检测其mRNA的表达水平。结果 TIP30基因在HCT116及HT29癌细胞中甲基化状态有差异,其表达水平与启动子CpG岛甲基化状态有关,并且二者对5-氟尿嘧啶敏感度不同。结论 TIP30基因启动子甲基化状态可能影响大肠癌细胞对化疗药物的敏感性,为大肠癌患者的个体化治疗提供了可能。     

Methylation of TIMP3 in esophageal squamous cell carcinoma

AIM： To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 （TIMP3） promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China. METHODS： Cancer cell lines were treated with or without the demethylating reagent 5-aza-2′-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry.
RESULTS： Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one.
CONCLUSION： Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.