MicroRNA profiling identifies miR-2355-5p as a key modulator in Renal Cell Carcinoma tumor growth

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is one of the first truncal event in clear cell Renal Cell Carcinoma (ccRCC) tumorigenesis. Accumulation of the Hypoxia Induced Factors (HIFα) acquired from VHL loss can promote ccRCC tumorigenesis through regulating microRNAs (miRNA) expression. Here, we performed a miRNA profiling and used high throughput analysis to identify a panel of VHL dependent miRNAs in ccRCC. Validation of these miRNAs uncovered overexpression of miR-2355-5p in ccRCC cell models, and primary tumors. Moreover, we showed a significant increase of circulating miR-2355-5p in plasma from patients with ccRCC. Mechanistically, miR-2355-5p overexpression was confirmed to be HIF-2α-dependent. Indeed, targeting miR-2355-5p with the CRISPR/cas9 system did not only negatively disturbed ccRCC cell’s ability to stimulate angiogenesis, but also decreased cell proliferation and drastically reduced tumor growth in mice xenograft models. Finally, miR-2355-5p pulldown assay identified five tumor suppressor genes, ACO1, BTG2, CMTM4, SLIT2, and WDFY2, as potential targets. All five genes were significantly downregulated in ccRCC tumors and mice xenograft tumors. Results from this research demonstrate the oncogenic ability of miR-2355-5p and shed light on the possible route in which the miRNA controls angiogenesis and tumor growth in VHL-deficient ccRCC To identify RNA targets of both miR-2355-5p and miR-1271-5p in ccRCC tumors, we performed a miRNA Pulldown assay in 786-0 cells. Biotinylated synthetic miRNA duplexes were transfected into 786-0 cells for 48h in triplicates. Biotinylated miRNA and bind RNA were retrieved and analyzed through sequencing platform. Enrich RNA transcripts were labeled as potential miRNA targets.