Abstract Background: Despite optimal and early surgical treatment of non-small-cell lung cancer (NSCLC), many patients die of recurrent NSCLC. In a recent study, we have undertaken an integrated genetic approach to discover novel loci and miRNA that are deregulated in NSCLC. Here we investigated the association between a panel of miRNA and clinicopathological parameters of the tumor. Methods: To study association between allelic imbalance/LOH and deregulation of miRNAs, we analyzed a subset (8 pairs) of samples for miRNA expression and correlate the miRNA expression profile with allelic imbalance data generated by SNP analysis. Two types of analysis (The average expression ratio for each miRNA and Mean tumor expression profile) were carried out to identify miRNA probes which show differential expression between tumor and adjacent normal tissues, and the findings were correlated with allelic imbalance area determined by SNP array analysis. A set of 8 miRNA (miR-21, miR-23b, miR-31, miR-126, miR-145, miR-150, miR-205, miR-296) were further analyzed in two independent cohorts of samples to determine their relationship with clinicopathological parameters including outcome. Each of the samples of these cohorts has at least 10 years of follow-up. Statistical analysis were performed the Mann-Whitney U-test, X2 test or Fisher's exact test, where appropriate. Overall survival rates were analyzed by the Kaplan-Meier and log-rank tests. The level of statistical significance was set at P < 0.05. Functional studies were performed for miR-23b. Results: We initially performed technical validation of 8 miRNAs by quantitative RT-PCR (Q-RT-PCR). In general, miRNA expression pattern determined by microarray and Q-RT-PCR are consistent. To determine cut off values, we further analyzed all the 8 miRNAs in 19 tumor samples with adjacent normal. By an optimal cut point, miR-21, miR-23b and miR 31 were found to be overexpressed in tumor when compared to paired normal, while miR-145 and MiR-150 were down-regulated. Using the same cut off point, we then analyzed an additional independent cohort of 127 early stage NSCLC samples and miR-23b and miR-31 were found to be overexpressed. Kaplan-Meier analysis indicated that patients with high miR-23b and miR 31 expression had a poor overall survival in univariate analysis (P=0.007, 0.012). Most importantly, multivariate analysis showed that miR-23b was an independent significant prognostic factor in patients with NSCLC (p=0.032). Overexpression of miR-23b facilitates NSCLC cell proliferation in vitro while inhibition of miR-23b decreases the cell viability. Conclusion: miR 23 b and miR-31 overexpression in patients with early stage NSCLC treated with curative intent by means of surgery is associated with poor survival. Larger independent confirmatory cohorts with longitudinal follow-up will be required in future studies to define the impact of these two miRNA overexpression in early stage NSCLC before clinical application. Citation Format: Begum Shahnaz, Masamichi Hayashi, Takenori Ogawa, Mariana Brait, William H. Westra, Mohammad O. Hoque, David Sidransky. Prognostic significance of miR-23b and miR-31 expression in non-small-cell lung cancer. [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 5231. doi:10.1158/1538-7445.AM2014-5231