Shen Wen-Jing, Dai Dong-Qiu, Teng Yue, Liu Hong-Bo
Department of Surgical Oncology, The First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China.
World J Gastroenterol. 2008 Jan 28;14(4):595-600. doi: 10.3748/wjg.14.595.
To investigate the changes of methylation state and expression of RASSF1A gene in human gastric cancer cell lines SGC7901 and BGC823 which were treated in vitro with demethlylating agent 5-Aza-CdR in combination with histone deacetylase inhibitor NaB.
After SGC7901 and BGC823 cells were treated with 5-Aza-CdR and/or NaB, the methylation state of RASSF1A gene was detected by methylation-specific PCR, and the changes in expression of mRNA and protein level of RASSF1A gene were observed by RT-PCR and Western-blotting before and after drug treatment.
Hypermethylation was detected in the promoter region of RASSF1A gene in both SGC7901 and BGC823 cells, and there was no expression of this gene at both mRNA and protein level. After treatment with 5-Aza-CdR, demethylation occurred in the promoter region of RASSF1A gene, which subsequently induced re-expression of this gene. The treatment with NaB alone showed no effect on the methylation state and expression of RASSF1A gene. The combined treatment of 5-Aza-CdR and NaB induced complete demethylation of RASSF1A gene, leading to a significantly higher re-expression of the mRNA and protein of RASSF1A than those treated with 5-Aza-CdR alone (P<0.05).
Hypermethylation in the promoter region is related to inactivation of RASSF1A gene in human gastric cancer cell lines SGC7901 and BGC823, while demethlylating agent 5-Aza-CdR can reverse the methylation state of RASSF1A gene and induce its re-expression. Histone deacetylase inhibitor NaB had a synergistic effect with 5-Aza-CdR in both demethylation and gene transcriptional regulation.
研究用去甲基化剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）联合组蛋白去乙酰化酶抑制剂丁酸钠（NaB）体外处理人胃癌细胞系SGC7901和BGC823后，RASSF1A基因甲基化状态及表达的变化。
用5-Aza-CdR和/或NaB处理SGC7901和BGC823细胞后，采用甲基化特异性PCR检测RASSF1A基因的甲基化状态，并用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western-blotting）观察药物处理前后RASSF1A基因mRNA和蛋白水平表达的变化。
SGC7901和BGC823细胞中RASSF1A基因启动子区域均检测到高甲基化，该基因在mRNA和蛋白水平均无表达。用5-Aza-CdR处理后，RASSF1A基因启动子区域发生去甲基化，随后该基因重新表达。单独用NaB处理对RASSF1A基因的甲基化状态及表达无影响。5-Aza-CdR与NaB联合处理诱导RASSF1A基因完全去甲基化，导致RASSF1A基因mRNA和蛋白的重新表达显著高于单独用5-Aza-CdR处理组（P<0.05）。
启动子区域高甲基化与人胃癌细胞系SGC7901和BGC823中RASSF1A基因失活有关，而去甲基化剂5-Aza-CdR可逆转RASSF1A基因的甲基化状态并诱导其重新表达。组蛋白去乙酰化酶抑制剂NaB在去甲基化和基因转录调控方面与5-Aza-CdR具有协同作用。
