Pleotrophic action of renal cell carcinoma: Dysregulated microRNAs on hypoxia-related signaling pathways

428 Background: The von Hippel-Lindau (VHL) gene is lost in 70% of clear cell Renal Cell Carcinomas (ccRCC); however, additional mechanisms are proposed to regulate VHL expression, including suppression by microRNAs (miRNAs). miRNAs are a class of naturally occurring, small non-coding RNA molecules that downregulate gene expression of target mRNAs. We demonstrate that ccRCC-dysregulated miRNAs can target multiple members of the ccRCC-related signaling pathways. Methods: miR-17 and miR-224 mimics and inhibitors were transfected into ccRCC cell lines using siPORT (Ambion). PicTar and TargetScan were used for target prediction. Target expression and miRNA expression was analyzed by qRT-PCR (Ambion). Western blot antibodies were purchased from Millipore or Cell Signaling. Cell lines were purchased from ATCC. All methods followed the manufacturer’s protocol. Results: According to our preliminary results, the miRNAs that are dysregulated in ccRCC specimens are predicted to target multiple members of the hypoxia-related pathways. To confirm the in silico analysis, miR-17 and miR-224 were selected for experimental target validation, as they were among the most up-regulated miRNAs in ccRCC. We experimentally validated VHL and HIF1α as likely direct targets of miR-17 and miR-224. Luciferase reporter assay confirmed that miR-17 directly downregulates VHL. Moreover, VHL protein level decreased upon miR-17 and miR-224 transfection. We also established a negative correlation between the expression of miR-17 and two predicted targets VEGF-A, EGLN3 in RCC specimens, and miR-224 and its predicted targets SMAD4 and SMAD5. This suggests that downstream signaling pathways are also modulated by miR-17 and miR-224. These results confirm the most important findings of the bioinformatics analysis: miR-17 targets different molecules along the same signaling pathway and that multiple ccRCC-dysregulated miRNAs can synergistically suppress a single target, which functions in the pathogenesis. Conclusions: Our results indicate that miRNAs possibly regulate hypoxia-related pathways at multiple points. This is of special interest as miRNAs may serve as potential therapeutic targets.