Abstract Background: Renal cell carcinoma (RCC) is among the ten leading cancer types in United States. The 5-year relative survival rate of regional or localized RCC is 65-92%, while that of metastatic RCC is only 12%. Tumor recurrence and metastasis represent two major obstacles in the successful treatment of cancer. Emerging lines of evidence suggest that cancer aggressiveness is associated with epithelial to mesenchymal transition (EMT). Therefore, it is of critical importance to regulate EMT and to develop effective therapeutic strategies for the treatment of recurrent and metastatic cancer. Critical regulators of EMT include transcription repressors and microRNAs that target key proteins involved in EMT. MicroRNAs are implicated in regulating cancer progression and metastasis. Here we show that miR-720 is positively associated with RCC by negatively regulating key metastasis suppressing genes. Methods: We performed a series of in vitro and in vivo experiments including qRT-PCR, FACS cell cycle and apoptosis, chemotactic transwell migration and invasion, immunoblotting and luciferase reporter assays along with intratumoral xenograft mouse model. Results: Elevated levels of miR-720 were observed in a panel of RCC cell lines and clinical tissues compared to non-malignant cell line and normal samples. Loss of miR-720 function inhibited proliferation, migration and invasion and induced apoptosis in RCC cell lines in vitro and repressed tumor growth in xenograft mouse model. Conversely, gain of miR-720 function in non-malignant HK-2 cells induced pro-cancerous characteristics. Silencing of miR-720 caused a marked induction in the levels of endogenous αE-catenin and E-cadherin protein levels in anti720 transfected cells compared to control. Whereas, miR-720 overexpression in RCC cell lines reduced activity of a luciferase reporter gene fused to the wild-type αE-catenin or E-cadherin 3' UTR compared to non-specific 3' UTR control indicating that αE-catenin-E-cadherin complex is a direct and functional target of miR-720 in RCC. We also observed attenuation of β-Catenin, CD44 and Akt expression in RCC cells transfected with miR-720 inhibitor compared to control. Further, miR-720 exhibited clinical significance in RCC. Expression of miR-720 significantly distinguished malignant from normal samples. Elevated miR-720 levels positively correlated with higher Fuhrman grade, pathological stage and poor overall survival of RCC patients. Conclusion: These findings uncover a new regulatory network in RCC involving metastasis-promoting miR-720 that directly targets expression of key metastasis-suppressing proteins E-cadherin and αE-catenin complex. These results suggest that therapeutic regulation of miR-720 may provide an opportunity to regulate EMT and metastasis in RCC. Citation Format: Nadeem S. Bhat, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Priyanka Kulkarni, Pritha Dasgupta, Z Laura Tabatabai, Guoren Deng, Rajvir Dahiya, Shahana Majid. Role of a prometastatic miRNA as a negative regulator of the key metastasis suppressor genes in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1111.