Fig 2: Gene regulatory network of TFs and functional enrichment analysis of DEOSGs. A, B Differentially TFs are shown in a volcano plot (A) and a heatmap (B). Green, red, and black dots represent genes expressed at relatively lower, higher, or equal levels. C A regulatory network comprising differentially expressed TFs and DEOSGs. Triangles represent TFs, and red and green indicate risk and protective factors, respectively. D The respective protein-protein interaction (PPI) network analysis of PML, PYCR1 and MELK from TCGA datasets. The colored dot indicates a straight functional protein cluster to the three key genes. (https://string-db.org/, http://gepia.cancer-pku.cn/index.html). E, F The GO and KEGG pathway enrichment analysis of PML, PYCR1 and MELK-related partners. Significantly enriched pathways are indicated in Y axis. Gene Ratio in the X axis represents the enrichment levels. The larger value of Gene Ratio represents the higher level of enrichment. KEGG, Kyoto encyclopedia of genes and genomes; GO, gene ontology

Fig 3: Clinical prognostic analysis and validation of hub genes. A Express levels of MELK, PYCR1, and PML in ccRCC tumor tissues or normal tissues in the TCGA database. B The overall survival curves of MELK, PYCR1, and PML according to the Kaplan–Meier Plotter database. C–E The association between the mRNA expression levels of MELK, PYCR1, PML and grade (C), stage (D), TNM stage (E)

Fig 4: Immune infiltration analysis of MELK, PML and PYCR1. A–C The correlation between MELK (A), PML (B), PYCR1 (C) and immune cell infiltration using the TIMER database. D Distribution of MELK, PYCR1, and MELK mRNA expression in diverse immune subtypes of ccRCC according to the TISIDB database

Fig 5: MELK and PYCR1 expression were elevated in ccRCC tissue by the tissue microarray. A, B The expression of MELK and PYCR1 in ccRCC tissues and adjacent normal tissues was detected by TMAs. C, D Representative views showed the IHC staining of normal tissues and tumor tissues including different clinical pathological stages. The enlarged images of MELK were selected from Figure A (normal corresponded to A10; Tumor T1a corresponded to D05; Tumor T1b corresponded to E09; Tumor T2 corresponded to F09). The enlarged images of PYCR1 were selected from Figure B (normal corresponded to A09; Tumor T1 corresponded to B04; Tumor T2 corresponded to F06; Tumor T3 corresponded to C06). Scale bar is 50 μm. E, F IHC score of MELK and PYCR1 expression showed the up-regulated levels in ccRCC tissues than benign renal tissues