Definition of MicroRNAs That Repress Expression of the Tumor Suppressor Gene FOXO1 in Endometrial Cancer

Endometrial cancer is the most common malignancy of the lower female reproductive tract. The tumor suppressor FOXO1 is downregulated in endometrial cancer compared with normal endometrium but the underlying mechanisms are not well understood. Using microRNA (miR) target prediction algorithms, we identified several miRs that potentially bind the 3'-untranslated region of FOXO1 transcripts. Expression profiling of normal and malignant endometrial samples by quantitative real-time PCR and Northern blot analysis revealed an inverse correlation between the levels of FOXO1 protein and the abundance of several of the in silico-predicted miRs, suggesting that loss of FOXO1 expression in endometrial cancer may be mediated by miRs. To determine the role of candidate miRs, we used the endometrial cancer cell lines HEC-1B and Ishikawa, which express FOXO1 at high and low levels, respectively. Expression of miR-9, miR-27, miR-96, miR-153, miR-182, miR-183, or miR-186, but not miR-29a, miR-128, miR-152, or miR-486 mimetics in HEC-1B cells was sufficient to significantly reduce the abundance of FOXO1. Conversely, FOXO1 expression was efficiently restored in the Ishikawa cell line upon simultaneous inhibition of miR-9, miR-27, miR-96, miR-153, miR-183, and miR-186. Moreover, induction of FOXO1 in Ishikawa cells by miR inhibitors was accompanied by G1 cell cycle arrest and cell death, and was attenuated by the small interfering RNA-mediated downregulation of FOXO1 expression. Our findings identify several miRs overexpressed in endometrial cancer that function in concert to repress FOXO1 expression. Further, aberrant miR expression results in deregulated cell cycle control and impaired apoptotic responses, and thus, may be central to endometrial tumorigenesis.