Fig. 3
KH-type splicing regulatory protein (KSRP) negatively regulates neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) to trigger the epithelial-mesenchymal transition (EMT) and promotes invasion of clear cell renal cell carcinoma (ccRCC) cells. A NEDD4L was overexpressed (left panel) and knocked-down (right panel) in Caki-1 cells as determined by Western blotting (WB). B Invasive abilities of Caki-1 cells overexpressing (left panel) or knocked-down (right panel) NEDD4L were determined by a Matrigel invasion assay. C, D Expression levels of NEDD4L and KSRP, and the invasive abilities were respectively determined by WB (C) and Matrigel invasion assays (D) in Caki-1 cells expressing shKSRP with or without co-expressing shNEDD4L as indicated. Data from D are shown as a percent of control cells. Values are presented as the mean ± SD of three independent experiments. ***p < 0.001, compared to the control group. ###p < 0.001, compared to KSRP shRNA-infected cells. E Gene set enrichment analysis of TCGA-KIRC patients with high (top 5%) versus low (bottom 5%) expression of KSRP or NEDD4L. A gene set of the EMT derived from HALLMARK was used. Normalized enrichment scores (NESs) and false discovery rates (FDRs) are shown in the enrichment plot. F Correlations of gene expressions of KSRP or NEDD4L with EMT markers are demonstrated in a correlation plot. RNA sequencing data of TCGA-KIRC patients (n = 510) were utilized to perform this analysis. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. G Caki-1 cells expressing either a KSRP shRNA, NEDD4L-expressing, or respective control vector and subjected to WB to determine expressions of EMT-related regulators. H Dot plots indicating NEDD4L substrates-enriched signaling. I NEDD4L-overexpressing and control Caki-1 cells were treated with or without 20 μM MG-132 for 4 h, and then the Akt, p-Akt, Snail, and GAPDH proteins were analyzed by WB. J All ccRCC patients from TCGA or E-MTAB-1980 were separated into a negative correlation with KSRP and NEDD4L expressions (low KSRP with high NEDD4L and high KSRP with low NEDD4L) and others. Data showed that patients in the KSRPhigh/NEDD4Llow group had the worst prognosis