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有研究表明,多个基因甲基化引起的表达改变可能是肿瘤对化疗药物敏感性的调控因素之一。为了寻找肺腺癌A549细胞对顺铂敏感性的生物标志,该研究以肺腺癌A549细胞及对顺铂耐受的同源A549细胞(A549-DDP)为研究对象,甲基化特异性PCR(MSP)检测五个候选基因(RASSFIA、MGMT、DAPK、p16、RARβ)的甲基化状态,RT-PCR检测候选基因在mRNA水平的表达。结果显示:在A549细胞中,RASSF1A、MGMT、p16呈低甲基化状态和高表达;顺铂耐受的A549DDP细胞中,这三个基因均以甲基化状态为主,mRNA表达明显下调;但RARβ仅在A549-DDP细胞中呈非甲基化和高表达,DAPK的甲基化和表达水平在两个细胞亚型间无明显差异。A549-DDP细胞经去甲基化试剂(5-aza-CdR)作用后,RT-PCR检测显示:RASSF1A、MGMT在A549-DDP细胞中的mRNA表达水平上调,并呈明显的剂量依赖和时间依赖关系。研究结果提示,RASSF1A、MGMT、pI6、rarβ基因甲基化修饰导致的mRNA表达改变可能是调控肺腺癌A549细胞对顺铂敏感性的因素之一。RASSF1A、MGMT、p16、RARβ甲基化谱作为肺癌对顺铂敏感性的生物标志,值得进一步研究。
Studies have shown that changes in the expression of multiple genes methylation may be one of the regulatory factors of tumor sensitivity to chemotherapeutic drugs. In order to find a biomarker of sensitivity to cisplatin in lung adenocarcinoma A549 cells, A549 cells (A549-DDP) and lung adenocarcinoma A549 cells, which are resistant to cisplatin, were studied. Methylation-specific PCR (MSP) were used to detect the methylation status of five candidate genes (RASSFIA, MGMT, DAPK, p16, RARβ). The mRNA expression of candidate genes was detected by RT-PCR. The results showed that RASSF1A, MGMT and p16 were hypomethylated and overexpressed in A549 cells. The methylation status and mRNA expression of all the three genes were down-regulated in cisplatin-resistant A549DDP cells. However, RARβ was only non-methylated and highly expressed in A549-DDP cells. The methylation and expression level of DAPK showed no significant difference between the two cell subtypes. After A549-DDP cells were treated with demethylation reagent (5-aza-CdR), the mRNA expression of RASSF1A and MGMT was up-regulated in A549-DDP cells in a dose-and time-dependent manner relationship. The results suggest that the changes of mRNA expression induced by methylation of RASSF1A, MGMT, pI6 and rarβ may be one of the factors that regulate the sensitivity of lung adenocarcinoma A549 cells to cisplatin. RASSF1A, MGMT, p16, RARβ methylation profile as a biomarker of sensitivity to cisplatin in lung cancer deserves further study.