microRNA expression is frequently altered in human cancers. Epigenetic silencing, especially DNA methylation, is one of many mechanisms of microRNA suppression in cancer. To search for epigenetically silenced microRNAs in non-small-cell lung cancer (NSCLC), we mapped human microRNAs on autosomal chromosomes and selected in silico 55 microRNAs that met one of the following criteria: (i) microRNAs within CpG islands, (ii) microRNAs within 1 kbp downstream of CpG islands, and (iii) microRNAs within gene introns whose promoters have CpG islands. 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 microRNAs. Fourteen microRNAs (mir-375, mir-196b, mir-126, mir-34b, mir-127, mir-203, mir-148a, mir-181c, mir-30e, mir-449a, mir-340, mir-486, mir-483, mir-139) were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. Among the 14 microRNAs, seven (mir-126, mir-34b, mir-203, mir-30e, mir-449a, mir-486, mir-139) were frequently suppressed in primary NSCLCs. 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, while 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. A significant enrichment of H3K27me3 was observed in lung cancer cell lines with decreased mir-126 expression, suggesting the involvement of a polycomb complex in the regulation of EGFL7 and mir-126. The overexpression of mir-34b and mir-126 by plasmid vectors 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 microRNAs 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.

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