DNA甲基化异常对mir-615-5p在胰腺癌细胞株PANC-1中表达影响

Abnormal DNA methylation in pancreatic cancer cell line PANC-1 affects mir-615-5 pexpression

目的:描述胰腺癌细胞株中DNA异常高甲基化对miRNA表达抑制所起的作用。观察DNA甲基转移酶抑制剂对miR-615-5p在胰腺癌细胞株PANC-1中甲基化状态和表达的影响。方法:甲基化芯片筛选出在胰腺癌细胞株PANC-1中存在异常甲基化的miRNA。采用去甲基化药物5-氮杂胞苷(5-aza-2-adeoxycytidine,5-aza-dC)处理体外培养的PANC-1细胞,甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)结合亚硫酸氢盐修饰结合测序法(bisulfite-sequencing PCR,BSP)检测用药前后miR-615-5p的甲基化状态的改变。采用RT-PCR定量检测用药前后miR-615-5p表达量的差异。结果:通过MSP和BSP发现在胰腺癌细胞株PANC-1中甲基化率明显高于正常组织。对胰腺癌细胞株用去甲基化剂5-aza-dC的作用,发现miR-615-5p甲基化率减少并伴随着表达的重新激活。结论:胰腺癌细胞株PANC-1中的miR-615-5p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miR-615-5p的表达沉默。

Objective： To observe the abnormal DNA methylation inhibits the miRNA expression in pancreatic cancer cell line,and DNA methyltransferase inhibitor on the miR-615-5p of the methylation status and expression in the pancreatic cancer cell linePANC-1. Methods： Methylation microarray was used to screen in pancreatic cancer cell line PANC-1 in the abnormal methylation of miRNA. PANC-1 cells were treated with demethylating agent 5-aza-2-adeoxycytidine （5-aza-dC） in vitro, Methylation specific poly-merase chain reaction （methyla-tion- specific PCR, MSP） combination of bisulfite modified DNA sequencing method （bisulfite-se-quencing PCR, BSP） were used to detect miR-615-5p methylation changes before and after treatment. The changes of miR-615-5 pexpression was detected by quantitative RT-PCR before and after treatment. Results： Methylation was significantly higher in pancre-atic cancer cell line PANC-1 than in normal tissue by MSP and BSP methods. miR-615-5p reduction in methylation was associatedwith the expression reactivation in the pancreatic cancer cells PANC-1 treated with demethylation agent 5-aza-dC. Conclusion： This study demonstrates that aberrant DNA methylation of the miR-615-5p CpG island is closely linked to their inappropriate silencing in pancreatic cancer cell line PANC-1.