Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA-135a (miR-135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR-135a and to identify miR-135a-mediated molecular pathways in RCC cells. Restoration of mature miR-135a significantly inhibited cancer cell proliferation and induced G 0 ⁄ G 1 arrest in the RCC cell lines caki2 and A498, suggesting that miR-135a functioned as a potential tumor suppressor. We then examined miR-135a-mediated molecular pathways using genome-wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR-135a transfected RCC cell lines. To investigate the biological significance of potential miR-135a-mediated pathways, we classified putative miR-135a-regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR-135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR-135a targeted the c-MYC gene in RCC. Gain-of-function and luciferase reporter assays showed that c-MYC was directly regulated by miR-135a in RCC cells. Furthermore, c-MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor-suppressive miR-135a-mediated molecular pathways could reveal potential therapeutic targets in RCC. (Cancer Sci 2013; 104: 304-312) R enal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and approximately 80% of RCC patients are diagnosed with the clear cell RCC (ccRCC) subtype.(1) In the USA, the incidence and mortality rates of RCC have increased in recent years, with approximately 58 000 new cases and 13 000 deaths in 2010.(2) Although surgery is curative for localized disease, a significant percentage of patients developed relapses or metastases with poor prognosis.(3,4) Therefore, researchers have become interested in elucidating the molecular mechanisms of RCC oncogenesis and metastasis, which could lead to development of better prognostic, diagnostic, and therapeutic interventions for the disease.RNA can be divided into two categories, protein-coding RNAs and non-coding RNAs (ncRNAs). It is important to examine the functions of ncRNAs in both normal and diseased tissues and to elucidate their involvement in human diseases, including cancer. MicroRNAs (miRNAs) are endogenous small ncRNA molecules (19-22 bases) that regulate protein-coding gene expression by repressing translation of RNA or cleaving RNA transcripts in a sequence-specific manner.(5) A growing body of evidence suggests that miRNAs are aberrantly expressed in many human cancers and ...