人胰腺癌细胞株MUC2基因启动子区甲基化状态检测和分析

Methylation Status of MUC2 Gene Promoters in Human Pancreatic Cancer Cell Lines

背景:近年表观遗传学修饰方式之一的DNA甲基化成为肿瘤研究的热点。目前已发现胰腺癌中存在MUC2表达异常。目的:探讨人胰腺癌细胞株MUC2表达与其基因启动子区甲基化的关系,以了解胰腺癌的发生机制。方法:以人胰腺癌细胞株AsPC-1、BxPC-3、CFPAC-1、PANC-1、SW1990和PaTu8988s为研究对象,以逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法检测去甲基化制剂DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)处理前后各胰腺癌细胞株MUC2 mRNA/蛋白表达的变化,以甲基化特异性PCR(MSP)结合测序检测MUC2基因启动子区CpG岛甲基化状态。结果:5-Aza-CdR处理前,胰腺癌细胞株AsPC-1、CFPAC-1和SW1990无MUC2 mRNA/蛋白表达或低表达:经5-Aza-CdR处理后,MUC2 mRNA/蛋白重新表达。MSP结合测序显示上述胰腺癌细胞株MUC2基因启动子区CpG岛存在高甲基化。结论:人胰腺癌细胞株MUC2表达抑制与其基因启动子区CpG岛高甲基化相关。MUC2基因启动子区CpG岛高甲基化可能在胰腺癌的发生、发展中起一定作用。

Background：DNA methylation is one of the modalities of epigenetic modification, and has drawn more attention in the studies of tumors in recent years. Studies have shown that there is aberrant expression of MUC2 mucin in pancreatic cancer. Aims： To explore the relationship between MUC2 expression and its promoter hypermethylation in human pancreatic cancer cell lines, so as to provide insights into the origins of pancreatic cancer. Methods： Human pancreatic cancer cell lines as AsPC-1, BxPC-3, CFPAC-1, PANC-1, SW1990 and PaTu8988s were collected. Reverse transcriptase polymerase chain reaction （RT-PCR） and immunocytochemistry were used to detect the expression of MUC2 at mRNA and protein levels in these cell lines before and after treatment with 5-aza-2＇-deoxycytidine （5-Aza-CdR）, a DNA methyltransferase inhibitor. Methylation-specific PCR （MSP） combined with DNA sequencing were adopted to identify the CpG island methylation of MUC2 gene promoters. Results： Expression of MUC2 mRNA/protein was absent or lower in pancreatic cancer cell lines AsPC-1, CFPAC-1 and SW1990 before 5-Aza-CdR treatment, and re-expressed after treatment with 5- Aza-CdR. CpG island hypenuethylation of MUC2 gene promoters was observed in AsPC-1, CFPAC-1 and SW1990 cells by MSP and DNA sequencing. Conclusions： Hypermethylation in CpG island of MUC2 gene promoters is correlated with the inhibition of MUC2 transcription in human pancreatic cancer cell lines, and might play an important role in the development and progression of pancreatic cancer.