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In this study, we examined the microRNA (miRNA) expression profile in laryngeal cancer. We further investigated the role of miR-133b and miR-375 in the growth of head and neck cancer (HNC) in vitro. METHODS: Expression profile of 723 human miRNAs in 8 laryngeal tissues (polyp, dysplasia, cancer, neighboring normal tissue matched to laryngeal cancer) was examined using microarrays. Subsequently, qRT-PCR was performed to confirm the microarray data using 48 laryngeal tissues including 16 laryngeal cancers on 3 miRNAs (miR-130b*, miR-133b, miR-375), which showed distinctive expression in cancer on microarray. Six cancer samples had matched laryngeal tissues next to the tumor. Pairwise Mann-Whitney's U test and Wilcoxon signed ranks test were utilized for statistical analyses. Furthermore, impact of miR-133b or miR-375 transfection on the growth of HNC cell lines was examined using WST-based assay. RESULTS: Microarray analysis revealed the up-regulation of 8 miRNAs, including miR-130b* and the down-regulation of 3 miRNAs, including miR-133b and miR-375. The results obtained from qRT-PCR were consistent with those obtained from microarray. Especially, the difference of miR-133b expression level between matched samples was significant (pϭ0.0277). MiR-133b suppressed the growth of SAS cell line and miR-375 suppressed the growth of JHU-011 cell line 5 days after transfection compared to control. CONCLUSIONS: We demonstrated the miRNA expression signature in laryngeal cancer and the role of miRNAs on the growth of HNC cell lines. Our novel findings may pave the way to elucidate the molecular mechanism of oncogenesis in HNC.

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Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, comprising roughly 6% of all malignancies worldwide, and laryngeal cancer has the highest incidence in it. At present, laryngeal cancer is treated with surgery and/or (chemo)radiation, each of which can have devastating consequences on speech and swallowing function. Even with the combined treatment approaches as mentioned above, laryngeal cancers with advanced stages have poor prognosis, therefore in need of novel less invasive treatments against their high morbidity. However, although to date its relevance to smoking has been well documented, the molecular mechanism involved in laryngeal cancer development and its highly sensitive biomarkers remain to be known. Thus, we have focused on microRNAs (miRNAs) showing highly tissue-or disease-specific patterns as potential novel biomarkers or therapeutic targets with clinical applicability. In this study, to screen the miRNAs highly sensitive to laryngeal cancer as putative biomarkers, we first examined the expression of 723 human miRNAs in 8 laryngeal tissues (1 polyp, 2 dysplasias, 3 laryngeal cancers and 2 neighboring normal tissues matched to laryngeal cancers) using microarrays (Agilent Human miRNA V2). The result showed the up-regulation of 6 miRNAs and the down-regulation of 3 miRNAs in laryngeal cancer compared to others. Subsequently, to confirm these findings and quantitate cancer-specific miRNAs, we performed quantitative RT-PCR (qRT-PCR) analysis (TaqMan miRNA assays, Applied Biosystems) using 48 laryngeal tissue samples including 16 cancers on 5 miRNAs (miR-196a, miR-130b*, miR-133b, miR-375, miR-455-5p), which showed markedly differential expression in cancers compared with others on microarrays. The results were consistent with those from microarrays, especially when cancer samples were compared with matched laryngeal tissues as normal controls. Of the 5 miRNAs examined, miR-196a showed significant increase in cancers when compared to either benign tissues or dysplasias. Next, to examine the utility of miR-196a as a promising biomarker for laryngeal cancer, we performed qRT-PCR on miR-196a using 83 tissue samples and 68 serum samples and found that miR-196a could be very useful in diagnosing the disease. We also analyzed miR-196a expression in FFPE sections by in situ hybridization and the result showed its robust expression in cancers but barely detectable in benign tissues Finally, to explore whether miR-196a can promote tumor growth in vitro or in vivo, we treated human laryngeal cancer cells or xenografted nude mice, respectively, with either miR-196a inhibitor or negative control inhibitor, and found that miR-196a inhibition suppresses tumorigenic properties of laryngeal cancer. Together with these findings, our study provided the first demonstration of miR-196a as a putative diagnostic biomarker and therapeutic target for laryngeal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4039.