Impact of aberrant methylation of microRNA-9 family members on non-small cell lung cancers

MicroRNAs (miRs) contribute to cancer development and progression by acting as oncogenes and tumor suppressor genes. miR-9 family members (miR-9s), including miR-9-1, 9-2 and 9-3, have been shown to be oncogenically involved through the downregulation of E-cadherin expression, which promotes the epithelial-mesenchymal transition. Tumor suppressive roles of miR-9s have also been reported to silence miR-9 through methylation, which is associated with an shortened overall survival (OS) period in several types of cancer. In this study, the impact of miR-9s methylation on non-small cell lung cancers (NSCLC) was investigated. In total, 293 resected NSCLC samples were examined and the miR-9s methylation status was determined using a combined bisulfite restriction analysis. miR-9 expression was analyzed by in situ hybridization. Methylation of miR-9-1, 9-2 and 9-3 was present in 20 (7%), 33 (11%) and 34 (12%) of the cases, respectively. Methylation of any miR-9s (miR-9s methylation) was observed in 76 of the cases (26%), and miR-9 expression was silenced in cases with miR-9s methylation. Logistic regression analysis demonstrated that male gender [odds ratio (OR), 2.0; 95% confidence interval (95% CI), 1.1–3.6; P=0.01] and pathologically negative lymph node metastasis (OR, 4.8; 95% CI, 1.4–17.2; P=0.002) were independent relative factors for miR-9s methylation. Additionally, miR-9s methylation [hazard ratio (HR), 4.2; 95% CI, 1.2–27.0; P=0.026] and early pathological stage (HR, 8.3; 95% CI, 2.1–28.6; P=0.004) were found to be independent predictive factors for prolonged OS time by the Cox proportional hazard test. miR-9s methylation which induces expression silencing is common in NSCLC cases without lymph nodal metastasis, suggesting that miR-9s are oncogenically involved in NSCLC carcinogenesis through the promotion of tumor metastasis.