Department of Respiratory Medicine, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, Japan.
Int J Cancer. 2012 Jun 1;130(11):2580-90. doi: 10.1002/ijc.26254. Epub 2011 Sep 17.
MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.
MicroRNA (miRNA) 的表达在人类癌症中经常发生改变。为了在非小细胞肺癌 (NSCLC) 中寻找被表观遗传沉默的 miRNA，我们在常染色体上绘制了人类 miRNA，并在计算机上选择了 55 个 miRNA。我们用 DNA 甲基化抑制剂 5-氮杂-2'-脱氧胞苷 (5-aza-CdR) 处理六株 NSCLC 细胞系，并测定了这 55 个 miRNA 的表达。有 14 个 miRNA 在癌细胞系中减少，并在 5-aza-CdR 处理后被诱导。经过详细的 DNA 甲基化分析，我们发现 mir-34b 和 mir-126 被 DNA 甲基化沉默。mir-34b 是由其自身启动子的 DNA 甲基化沉默的，而 mir-126 是由其宿主基因 EGFL7 的 DNA 甲基化沉默的。染色质免疫沉淀试验显示 mir-34b 和 EGFL7 中有 H3K9me2 和 H3K9me3，EGFL7 中有 H3K27me3。mir-34b 和 mir-126 的过表达分别降低了 c-Met 和 Crk 的表达。肺癌细胞系的 5-aza-CdR 处理导致 mir-34b 表达增加，c-Met 蛋白减少。接下来，我们使用 99 例原发性 NSCLC 分析了这些 miRNA 的 DNA 甲基化状态。mir-34b 和 mir-126 在所有病例中分别有 41%和 7%被甲基化。mir-34b 的 DNA 甲基化与免疫组化确定的 c-Met 表达无关，但在多变量分析中，mir-34b 甲基化（p=0.007）和 c-Met 表达（p=0.005）均与淋巴浸润显著相关。mir-34b 的 DNA 甲基化可作为肺癌侵袭表型的生物标志物。
