DNA甲基化调控肺腺癌微小RNA-145表达的分子机制

Molecular mechanism of microRNA-145 down-regulating expression by DNA methylation in lung adenocarcinoma

目的 观察肺腺癌组织中微小RNA（miRNA,miR）-145基因启动子区域各个胞嘧啶-磷酸-鸟嘌呤基序（CpG）岛的甲基化情况,探讨DNA甲基化在肺腺癌中调控miR-145表达的分子机制.方法 用DNA甲基化转移酶抑制剂5-氮杂胞苷（5-aza-C）对肺腺癌细胞株A549、H1975进行处理,比较miR-145的表达变化.收集手术切除的肺腺癌及癌旁组织标本共20对,应用甲基化DNA定量分析平台和半定量反转录-聚合酶链反应（RT-PCR）技术,对癌和癌旁组织miR-145启动子区域的甲基化水平进行定量分析.结果 经5-aza-C处理后,A549和H1975细胞株中的miR-145表达均明显上调.甲基化定量数据显示在肺腺癌组织miR-145基因启动子区域的甲基化率均值明显高于癌旁组织（0.59比0.32,P＜0.01）.并且在肺腺癌组织中,miR-145基因启动子区域的所有13个CpG位点均呈显著高甲基化：CpG1：13.55±2.06比19.30±5.83;CpG2：27.65±5.68比42.85±15.64;CpG3：36.40 ±6.28比60.20±17.55;CpG4：28.40±6.45比42.25±16.78;CpG5、6：28.65±3.91比41.50±10.19;CpG7：14.40±3.14比27.75±11.69;CpG8、9、10：55.50±4.85比76.50±8.52;CpG11：48.10 ±3.51比64.20±6.26;CpG12：52.75±5.90比73.35±12.70;CpG13：45.80±4.71比58.70±15.40.结论 肺腺癌细胞内miR-145表达受其基因启动子区甲基化调控;肺腺癌组织内miR-145基因启动子区CpG岛高甲基化与该基因表达水平变化密切相关.

Objective To investigate the epigenetic mechanisms of microRNA （miRNA,miR）-145expression in lung adenocarcinoma by detection of miR-145 gene methylation levels at the promoter region.Methods We treated lung adenocarcinoma cell lines A549 and H1975 with DNA methyltransferase inhibitors 5-azacytidine,and compared the level of miR-145 before and after the treatment.Then we collected 20 pairs of human lung adenocarcinoma tissues and the matched adjacent normal tissues,and analyzed the methylation level of cytosine-phosphoric acid-guanine motif （CpG） sites at the promoter region of miR-145 by using the Sequenom MassARRAY platform and reverse transcriptase-polymerase chain reaction（RT-PCR）.Results The expression of miR-145 was elevated significantly after the treatment with 5-azacytidine.The average level of methylation at the promoter region of miR-145 was remarkably higher in the lung adenocarcinoma than in the normal control （0.59 vs.0.32,P 〈 0.01）.Specifically,the methylation level for each of the 13 CpG site in lung adenocarcinoma was dramatically higher：CpG1：13.55±2.06 vs.19.30±5.83,CpG2：27.65 ±5.68 vs.42.85±15.64,CpG3：36.40 ±6.28 vs.60.20 ± 17.55,CpG4：28.40 ± 6.45 vs.42.25 ± 16.78,CpG5,6：28.65 ± 3.91 vs.41.50 ± 10.19,CpG7：14.40 ±3.14 vs.27.75 ± 11.69,CpG8,9,10：55.50 ±4.85 vs.76.50 ±8.52,CpG11：48.10 ± 3.51 vs.64.20 ± 6.26,CpG12：52.75 ± 5.90 vs.73.35 ± 12.70,CpG13：45.80 ± 4.71 vs.58.70 ± 15.40.Conclusion Our results revealed that methylation of the promoter region might contribute to the down-regulated expression of miR-145 in lung adenocarcinoma.