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目的:检测Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)在胰腺癌细胞株中的甲基化和表达状态,探讨其启动子异常甲基化在胰腺癌发病过程中的作用。方法:采用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联合TA克隆测序检测胰腺癌细胞株PANC-1及胰腺癌组织、癌旁组织及正常胰腺组织中RASSF1A启动子区CpG岛的甲基化状态,以甲基化酶抑制剂5-aza-2-deoxycitydine(5-aza-dC)处理PANC-1,观察处理前后甲基化率变化情况,逆转录PCR观察RASSF1A的mRNA表达情况。结果:在PANC-1细胞中RASSF1A启动子的甲基化率平均为100.00%,在正常胰腺、癌旁及癌组织中平均分别为1.79%、93.75%和100.00%,与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高(P<0.01),而癌旁及癌组织之间无明显差异(P>0.05)。在PANC-1细胞、胰腺癌组织及癌旁组织中RASSF1A基因无表达,在正常胰腺组织中RASSF1A基因呈阳性表达;PANC-1细胞经5-aza-dC处理后,RASSF1A的甲基化率下降(88.89%,P<0.05),mRNA表达无变化。结论:胰腺癌细胞株PANC-1及癌组织、癌旁组织RASSF1A基因表达与启动子区甲基化状态有关,启动子区的高甲基化导致PANC-1中RASSF1A基因的表达沉默。该基因异常甲基化有望成为胰腺癌的早期诊断指标和治疗靶点。
AIM: To detect the methylation and expression of Ras association domain family 1A (RASSF1A) in pancreatic cancer cell lines and to explore the role of promoter methylation in the pathogenesis of pancreatic cancer. Methods: The pancreatic cancer cell line PANC-1 and the pancreatic cancer tissues, adjacent non-cancerous tissues and normal pancreatic tissues were examined by bisulfite genomic sequencing PCR (BSP) combined with TA cloning sequencing. PANC-1 was treated with methylase inhibitor 5-aza-2-deoxycitydine (5-aza-dC), and the changes of methylation rate before and after treatment were observed. The mRNA expression of RASSF1A was observed by reverse transcription polymerase chain reaction. Results: The average rate of methylation of RASSF1A promoter in PANC-1 cells was 100.00%, and it was 1.79%, 93.75% and 100.00% respectively in normal pancreatic tissues, paracancer tissues and cancer tissues. Compared with normal pancreatic tissues, The methylation rate of RASSF1A promoter in adjacent tissues and cancerous tissues was significantly increased (P <0.01), but there was no significant difference between adjacent and cancerous tissues (P> 0.05). RASSF1A gene was not expressed in PANC-1 cells, pancreatic cancer tissues and paracancerous tissues, and RASSF1A gene was expressed in normal pancreatic tissues. The methylation of RASSF1A was decreased in PANC-1 cells treated with 5-aza-dC (88.89%, P <0.05), no change in mRNA expression. CONCLUSION: The expression of RASSF1A gene in pancreatic cancer cell line PANC-1, adjacent tissues and adjacent tissues is related to the methylation status of promoter region. The hypermethylation of promoter region leads to the silencing of RASSF1A gene expression in PANC-1. Aberrant methylation of this gene is expected to be an early diagnostic and therapeutic target for pancreatic cancer.