Abstract Introduction: MicroRNAs (miRs) are ∼22-nucleotide, non-coding RNAs that regulate gene expression post-transcriptionally. Recent data have indicated that miRs are aberrantly expressed in almost all human malignancies, including cervical cancer. In order to better understand how miRs might mediate tumor progression in cervical cancer, dysregulated miRs were identified and functional studies performed for miR-196b. Experimental Procedures: miR expression was measured in 3 cervical cancer cell lines (SiHa, ME-180 and HT3) and 3 normal cervix tissues using a quantitative real-time PCR approach examining 365 miRs and 3 endogenous controls. SiHa cells were transfected with Pre-miR miRNA Precursor Molecules (Ambion) to determine the effects of miR-196b overexpression. Candidate miR-196b targets were elucidated using a tri-pronged approach combining in silico target prediction algorithms, clinical mRNA expression data, and in vitro mRNA expression data following transfection of cell lines. To test for direct binding of miR-196b to the HOXB7 3’ UTR, a luciferase reporter assay was performed. Reporter vectors were constructed containing either the HOXB7 3’ UTR sequence (pMIR-REPORT-HOXB7/UTR), or the HOXB7 3’ UTR sequence with a mutation in the predicted miR-196b binding site (pMIR-REPORT-HOXB7/mut). SiHa cells were serially transfected with: a) miR-196b pre-miR or negative control pre-miR, and b) pMIR-HOXB7/UTR or pMIR-HOXB7/mut, and luciferase activity was measured 24 hours later. To determine the effect of knocking down HOXB7, SiHa cells were transfected with siHOXB7 (Qiagen). Results: Twenty-seven miRs were observed to be downregulated in all three cell lines compared to normal cervix. One of the 27 downregulated miRs, miR-196b, was shown to inhibit cell growth and colony-forming ability when overexpressed in SiHa cells. HOXB7 was identified as one of the candidate targets of miR-196b using our tri-pronged approach. Knocking down HOXB7 recapitulated the inhibition in cell growth and clonogenicity that was observed following mir-196b overexpression. When the luciferase reporter assay was performed, miR-196b overexpression resulted in a significant reduction in luciferase activity in cells transfected with pMIR-HOXB7/UTR, but not in cells transfected with pMIR-HOXB7/mut, demonstrating direct and specific binding of miR-196b to the 3’ UTR of HOXB7. Conclusion: miR-196b is downregulated in cervical cancer and negatively regulates HOXB7. 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 2059.