Background and objective: It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The 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-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D). DNA sequencing was used to confirm the methylation results.
Results: The level of miR-182 expression significantly differs among these lung cancer cell lines. The 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-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present 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 significantly increases when treated with 10 μM 5'-Aza-dC.
Conclusions: DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This 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 occurrence and development. .
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-dC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-dC）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。.