肺癌细胞中miR-182启动子甲基化状态研究

Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines

背景与目的已有的研究证明MiR-182的异常调控与恶性肿瘤的发生发展密切相关,本研究旨在探讨肺癌细胞中miR-182启动子的甲基化状态对miR-182表达的影响。方法荧光定量PCR检测肺癌细胞中miR-182表达水平,甲基化特异性PCR检测各细胞株中miR-182启动子区的甲基化状态,并通过测序进行验证。DNA甲基转移酶抑制剂5’-Aza-d C处理后检测各肺癌细胞株中miR-182表达变化。结果 MiR-182在不同肺癌细胞株的表达水平不同,其中,在高转移性肺癌细胞株如A549和L9981中相对呈低表达;而在低转移性细胞株95C则相对呈高表达。MSP及测序分析显示多株肺癌细胞株中miR-182启动子区域存在DNA甲基化,其中A549细胞甲基化程度最高。在5'-氮杂-脱氧胞苷酸(5’-Aza-d C)作用下,A549细胞及其他肺癌细胞中miR-182表达水平均明显升高。结论在肺癌细胞中miR-182启动子区域存在异常甲基化,miR-182的表达受DNA甲基化的调控。MiR-182的甲基化在肺癌中的作用尚需进一步研究。

Background and objective It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. hTe aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. Methods Real-time quantitative PCR and methylation-speciifc PCR were used to detect the expression level of miR-182 and its promoter methylation status in ifve lung cancer cell lines （A549, L9981, NL9980, 95C and 95D）. DNA sequencing was used to conifrm the methylation results. Results hTe level of miR-182 expression signiifcantly differs among these lung cancer cell lines. hTe highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-speciifc PCR and DNA sequencing assay results indicate that these lung cancer cell lines pres-ent different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines signiifcantly increases when treated with 10μM 5’-Aza-dC. Conclusion DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. hTis methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occur-rence and development.