Methylation of microRNA-9 is a specific and sensitive biomarker for oral and oropharyngeal squamous cell carcinomas.

Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.