Our recent studies of microRNA (miRNA) expression signatures have indicated that the miR-143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in cancer cells and to identify novel molecular targets of the miR-143/145 cluster in renal cell carcinoma (RCC). The expression levels of miR-143 and miR-145 were significantly downregulated in RCC tissues compared with adjacent non-cancerous tissues. A significant positive correlation was recognized between miR-143 and miR-145 expression. Restoration of mature miR-143 or miR-145 in 786-O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR-143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis showed that the hexokinase-2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells, was a candidate target of the miR-143/145 cluster. Luciferase reporter assays showed that both miR-143 and miR-145 directly regulated HK2. In RCC clinical specimens, the expression of HK2 was significantly higher in cancer tissues than in non-cancerous tissues. Silencing HK2 suppressed RCC cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. Thus, our data showed that loss of the tumor-suppressive miR-143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2. (Cancer Sci 2013; 104: 1567-1574 R enal cell carcinoma (RCC) is a disease in which cancer cells form in the tubules of the kidney. Globally, the incidence and mortality rates of RCC are increasing 2-3% per decade.(1) The 5-year survival rate of advanced-stage RCC is very poor (5-10%) due to recurrence or distant metastasis.(2)The latest treatment for RCC includes molecular targeted therapies, which have been developed and are being widely used for patients with metastatic or recurrent RCC. However, these types of therapies are not expected to have curative effects. Therefore, to improve outcomes in patients with RCC, it is necessary to fully elucidate the molecular mechanisms of RCC.The discovery of non-coding RNAs (ncRNAs) in the human genome was an important conceptual breakthrough in the postgenome sequencing era, (4) and it is now evident that improved understanding of ncRNAs is necessary for continued progress in cancer research. MicroRNAs (miRNAs) are endogenous small ncRNA molecules (19-22 bases in length) that regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. miRNAs are unique in their ability to regulate multiple protein-coding genes and are predicted to regulate more than 60% of the protein-coding genes in the human genome. (6) A growing body of evidence suggests that miRNAs are aberrantly expressed in many human cancers and that they play signif...