Pleotrophic Action of Renal Cell Carcinoma-Dysregulated microRNAs on Hypoxia Related Signaling Pathways
Zsuzsanna Lichner, Salvador Mejia-Guerrero, Monika Ignacak, Adriana Krizova, Tian Bao, Adrew Girgis, Yousef Youssef, George M Youssef. St Michael's Hospital, Toronto, ON, Canada
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.
Design: 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. RNA was isolated by miRNAeasy Kit (Qiagen), target expression was analyzed by qRT-PCR (Ambion). miRNAs were quantified by TaqMan assay (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.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 270, Wednesday Morning