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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 describe the role of abnormal DNA hypermethylation in miRNA expression in pancreatic cancer cell lines. To observe the effect of DNA methyltransferase inhibitor on the methylation status and expression of miR-615-5p in pancreatic cancer cell line PANC-1. Methods: The methylated microarray was used to screen out the abnormally methylated miRNA in pancreatic cancer cell line PANC-1. PANC-1 cells cultured in vitro were treated with 5-aza-2-adeoxycytidine (5-aza-dC), and methylation-specific PCR , MSP) combined with bisulfite-sequencing PCR (BSP) before and after treatment of miR-615-5p changes in methylation status. The difference of miR-615-5p expression before and after treatment was quantitatively determined by RT-PCR. Results: The methylation rate of pancreatic cancer cell line PANC-1 was significantly higher than that of normal tissues by MSP and BSP. The effect of demethylating agent 5-aza-dC on pancreatic cancer cell lines showed that the methylation rate of miR-615-5p was reduced and accompanied by the reactivation of the expression. Conclusion: The expression of miR-615-5p in pancreatic cancer cell line PANC-1 is related to the methylation status of promoter region. The hypermethylation of promoter region results in the silencing of miR-615-5p expression in PANC-1.