Fig. 1

Profiling of VHL-regulated miRNAs in ccRCC. A Heatmap showing significant DE miRNAs in RCC4 cells based on VHL status. MiRNAs with an absolute FC ≥ 1.5 and an FDR ≤ 0.05 were considered significantly DE. Bold miRNAs are also significantly DE in ccRCC tumors (B-C). B-C Volcano plot summarizing the miRNA expression profiles of ccRCC patients obtained from the TCGA-KIRC (B) and CPTAC3 (C) databases. Only patients whose miRNA sequencing was performed on both paired primary tumors and normal adjacent tissues were considered (TCGA-KIRC: N = 71, CPTAC3: N = 148). Significant miRNAs (red) were chosen as described in A. D Schematic representation of the similar miRNAs identified between analyses. The blue circles represent the number of miRNAs selected for further analysis. E Immunoblot analysis of VHL, HIF-1α, HIF-2α, and β-actin in RCC4, RCC10, and 786-0 cell lines with or without VHL. β-actin and total protein were used as loading controls. F RT-qPCR quantification of selected miRNAs in the cell lines described in E (N = 3–4). G Expression of selected miRNAs in ccRCC patients (CPTAC3) based on tumor VHL status. H-I Paired expression of miR-2355-5p in ccRCC patients from the TCGA-KIRC (H) and CPTAC3 (I) databases. MiR-2355-5p was deemed upregulated when \(\:\frac{Tumor\:CPM}{Paired\:Normal\:Tissue\:CPM}>1.5\). J RT-qPCR quantification of miR-2355-5p in ccRCC primary tissue compared to normal adjacent tissue (N = 13). K RT-qPCR quantification of miR-2355-5p in plasma from ccRCC patients (N = 43) compared to plasma from healthy individuals (N = 12). MiRNA expression was normalized with RNU44 (F), RNU48 (J) or Cel-miR-39-3p spike-in (K). The data are presented as the Mean ± SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test (F), Wilcoxon signed-rank tests (G, H, I), or Mann-Whitney U tests (J, K). (*P < 0.05, **P < 0.01, ***P < 0.001).