Abstract Head and neck squamous cell carcinomas (HNSCC) have poor prognosis due in part to diagnosis in advanced stages. Also, little progress on treatment modalities has been observed in the last decades. Identification of early detection molecular markers and new targets for therapy can bring important benefits for HNSCC patients’ survival. Recently, inactivating mutations in the NOTCH1 gene were described as the most frequent after TP53, suggesting an important role of this gene as a tumor suppressor in HNSCC. Another important alteration for HNSCC tumorigenesis is the de-regulated expression of microRNAs (miRs), small molecules known to negatively regulate their target mRNAs. Many studies trying to understand the biogenesis and mechanisms of miRs on gene expression regulation have proved this class of nucleic acids to be a good target for the identification of new biomarkers. In this study, we evaluate possible interactions between miR and the Notch pathway in HNSCC. To identify differentially expressed genes from Notch pathway and those miRs predicted to target components from this pathway, we performed mRNA and miR expression profiling of 44 HNSCC samples and 25 normal samples. Expression patterns were then validated in an independent cohort of 55 HNSCC tumors and 18 normal samples, using qRT-PCR. Microarray data analysis showed significant altered expression of 3 Notch pathway components: up-regulation of JAG1 and MAML1, and down-regulation of NUMBL. After identification of differentially expressed miRs, using online prediction tools, we selected the miRs expected to regulate these genes: miR-23a (MAML1), miR-26a (JAG1, MAML1), miR-146a (NUMBL), and let-7g (NUMBL). All four miRs were down-regulated in the 20 HN cell lines evaluated. Expression analysis in the independent cohort confirmed up-regulation of JAG1 in tumors. miRs expression evaluation confirmed miR-23a up-regulation in HNSCC samples. Forced expression of miR-23a (mimic) resulted in reduced cell proliferation in HN cancer cell lines. Our results suggest that miR-23a plays an important role in HNSCC and may be participating in Notch pathway regulation. To assess this interaction, mRNA expression analysis of members of Notch pathway before and after miR-23a overexpression on the cell lines is ongoing. We are also underway of comparing all the molecular changes observed in the HNSCC samples to demographic and clinicopathological variables to further evaluate Notch pathway alterations as well as miRs as potential HNSCC molecular markers. Citation Format: Myriam Loyo, Luciane T. Kagohara, Wenyue Sun, Marietta Tan, Elana Fertig, Michael Ochs, Evgeny Izumchenko, Mariana Brait, Joseph Califano, David Sidransky. Analysis of the interaction between microRNAs and Notch pathway members in HNSCC (head and neck squamous cell carcinoma). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 527. doi:10.1158/1538-7445.AM2014-527