Chen Hong, Pan Ying, Cheng Zheng-Yuan, Wang Zhi, Liu Yang, Zhao Zhu-Jiang, Fan Hong
Department of Gastroenterology, Affiliated Zhongda Hospital, Southeast University, Nanjing, China E-mail :
Asian Pac J Cancer Prev. 2013;14(11):6261-5. doi: 10.7314/apjcp.2013.14.11.6261.
Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer.
Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting.
The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR.
Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.
近期研究表明，RAS蛋白激活剂样1基因（RASAL1）在胃癌组织和细胞系中的表达降低，提示其在胃癌发生发展中发挥作用。RASAL1表达降低可导致癌细胞中RAS信号通路活性异常增加。然而，导致RASAL1基因下调的确切机制仍不清楚。本研究旨在确定RASAL1在胃癌中的甲基化状态及其调控机制。
采用甲基化特异性聚合酶链反应（MSP），评估40例患者胃癌组织及配对的癌旁非癌组织中RASAL1启动子区CpG岛的甲基化状态，并分析其临床病理意义。在用DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理后，还测定了胃癌细胞系MKN-28、SGC-790l、BGC-823以及正常胃上皮细胞系GES-1中RASAL1的甲基化状态。通过下拉法评估RAS活性（GAS-GTP），同时通过蛋白质印迹法测定RAS信号通路下游分子ERK1/2的蛋白水平。
胃癌组织及配对的癌旁非癌组织中RASAL1启动子甲基化频率分别为70%（28/40）和30%（12/40）（P<0.05）。胃癌患者中，RASAL1启动子甲基化与肿瘤分化、肿瘤大小、浸润深度及淋巴结转移之间存在显著相关性（均P<0.05），但与年龄或性别无关。在MKN-28、SGC-790l和BGC-823癌细胞中检测到RASAL1基因启动子高甲基化，而在正常胃上皮细胞系GES-1中未检测到。用5-Aza-CdR处理后，在三种胃癌细胞系中检测到RASAL1蛋白表达升高、RAS-GTP降低以及p-ERK1/2蛋白降低。
RASAL1基因启动子异常高甲基化在胃癌组织和细胞中频繁发生。此外，去甲基化剂5-Aza-CdR可逆转RASAL1基因的高甲基化，并在体内显著上调胃癌细胞中RASAL1的表达。我们的研究表明，RASAL1启动子甲基化可能与胃癌中RASAL1表达降低有一定关系。
