Abstract Commonly observed in colorectal cancer (CRC) is elevated expression of the prostaglandin synthase cyclooxygenase-2 (COX-2). In normal intestinal epithelium, the COX-2 mRNA is normally targeted for rapid decay through RNA elements present within its 3′-untranslated region (3′UTR), whereas in tumors rapid mRNA decay is compromised. To understand the mechanisms controlling COX-2 mRNA fate, we show that the COX-2 3′UTR can mediate mRNA degradation through microRNA-mediated regulation. MicroRNAs (miRNAs) are small non-coding RNAs that bind within the 3′UTR of target mRNAs and function as post-transcriptional regulators of gene expression. MiR-16 was identified to bind the COX-2 3′UTR and inhibit COX-2 expression by promoting rapid mRNA decay. Given the importance of miR-16 in cancer development, we examined miR-16 expression in CRC tumor specimens and cancer cells and observed a 2-fold decrease in miR-16 levels. Additionally, expression of miR-16 in cancer cells attenuated COX-2 expression and prostaglandin synthesis. The in vivo role of miR-16 in the initiation and development of colorectal cancer was examined using ApcMin/+ mice, a mouse model of intestinal neoplasia. To accomplish this, endogenous miR-16 was inhibited in the GI tract using an anti-miR oligonucleotide (AMO) against miR-16 containing 2′-O-(2-Methoxyethyl) modification with unmodified PO backbone. 50 mg/kg of AMO-16 or control AMO containing 4 nucleotide-mismatch of the AMO miR-16 sequence were administered by oral gavage to 8 week old ApcMin/+ and C57Bl/6 control mice twice a week over a 3-week period. Specific inhibition of miR-16 in intestinal tissue was observed. Over the treatment course, control mice displayed normal growth characteristics. Gross and histological analysis did not reveal any morphological changes in the GI tract of AMO-16-treated C57Bl/6 mice. In contrast, a significant 2-fold increase in small intestinal tumor burden was observed in ApcMin/+ mice treated with AMO-16 compared to control AMO (55.7 +/− 5.0 vs. 99.1 +/− 7.9, P = 0.0001). Gene expression analysis showed increased COX-2 and several miR-16 target cell cycle regulatory genes including cyclin D1, cyclin D3, cyclin E1, and CDK6 in AMO-16-treated small intestinal tissue from ApcMin/+ mice compared with AMO control treated mice, with the greatest level of altered expression observed in the proximal portion of the small intestine. These results identify miR-16 as a central post-transcriptional regulator of COX-2 along with other transcripts associated with promoting cancer cell proliferation, and reveal miR-16 to have tumor-suppressor properties in GI tumorigenesis downstream of a tumor-initiating event. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2949. doi:1538-7445.AM2012-2949