Abstract 2311: microRNA-196b regulates HOXB7 in cervical cancer
Authors: Christine How, Angela Hui, Paul Boutros, Blaise Clarke, Melania Pintilie, Anthony Fyles, Fei-Fei Liu
Journal: Cancer Research (2012)

Abstract Introduction: microRNAs (miRNAs) have been shown to play an important biological role in many human malignancies. The down-regulation of miR-196b has been previously reported in cervical cancer (CaCx), but its contribution to tumor progression remains unelucidated. Materials and Methods: miRNA expression was measured in frozen tissues from CaCx biopsies (n=79) and normal cervix (n=11), plus three CaCx cell lines (ME-180, SiHa and HT-3) using a quantitative real-time PCR (qPCR) approach simultaneously measuring 377 miRNAs. SiHa and ME-180 cells were transfected with 30 nmol/L of pre-miR-196b, pre-miR Negative Control (NC), siHOXB7, siVEGF, or Negative Control siRNA. Cell viability, clonogenicity and migration/invasion were analyzed using the Trypan blue exclusion assay, clonogenic assay and trans-well migration assays, respectively. Protein levels of HOXB7 and VEGF were measured by immunoblotting and ELISA, respectively. To determine candidate mRNA targets of miR-196b, a tri-modal approach was utilized by combining: a) all predicted targets from five target prediction databases; b) genes upregulated in CaCx patients; and c) genes down-regulated after in vitro miR-196b over-expression. A luciferase reporter assay was used to confirm the binding of miR-196b to the HOXB7 3′ UTR. Tumor formation was monitored in xenograft tumors in SCID mice; CD31 and Ki67 immunostaining were performed on tumors 25 days after implantation. Results: Significant down-regulation of miR-196b was observed in the CaCx cells lines and tissues. Patients with low miR-196b expression (n=39) experienced worse disease-free survival compared to those with high (n=39) miR-196b expression (p=0.02, HR=0.39). Pre-miR-196b transfection reduced cell viability (25% after 48h; 41% after 72h), clonogenicity (57%) and invasion (32% vs. 63% for cells treated with NC). In vivo, pre-miR-196b slightly decreased tumor growth, but associated with significantly reduced CD31 (69%) and Ki-67 expression (46% vs. 53% for NC cells). The luciferase reporter assay verified that HOXB7 is a direct and specific target of miR-196b. Decreased VEGF mRNA (43%) and protein (78%) levels after HOXB7 knockdown demonstrated that VEGF was a downstream mediator of HOXB7 activation. siRNA knockdown of HOXB7 or VEGF recapitulated the biological effects of miR-196b over-expression, including reduced cell viability (66% for siHOXB7 at 72h; 60% for VEGF at 72h), clonogenicity (69% for siHOXB7; 78% for siVEGF) and invasion (52% for siHOXB7 and 42% for siVEGF vs. 74% for Negative Control cells); thus corroborating the miR-196b/HOXB7/VEGF axis in CaCx progression. Conclusion: We have newly identified the miR-196b/HOXB7/VEGF pathway as an important dysregulated axis contributing to human CaCx progression; hence therapeutic targeting of this pathway could potentially improve patient outcome. 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 2311. doi:1538-7445.AM2012-2311