Fig. 5

KH-type splicing regulatory protein (KSRP) negatively regulates neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) mRNA stability via inducing miR-629-5p upregulation and targeting AU-rich elements (AREs) of the 3' untranslated region (3’UTR). A Decay rate of NEDD4L mRNA after treatment with 5 μg/mL actinomycin D for the indicated times in Caki-1 cells with or without KSRP-knockdown. B Flowchart for identifying miRNA induced by KSRP. The left part shows that TCGA miRNA sequencing data were retrieved from UCSC Xena. TCGA KIRC patients with low expression of miRNA (> 10% patients with reads per million mapped reads (RPM) of < 1) were excluded. The remaining 401 miRNAs were used to perform a differentially expressed (DE)-miRNA analysis between tumor (n = 241) and normal tissues (n = 70). Forty miRNAs were identified as upregulated miRNAs (with fold change (FC) of > 2 and a false discovery rate (FDR) of < 0.01) in KIRC tumor tissues. Right part shows 29 miRNAs with an FC of < 0.5 as identified in KSRP-depleted Caki-1 cells. By intersecting miRNAs upregulated in TCGA KIRC tissues with miRNAs induced by KSRP in ccRCC cells, miR-629-5p was identified as a critical candidate. C Real-time qPCR analysis of miR-629-5p in Caki-1 cells transfected with a KSRP shRNA, pEGFP-KSRP, or their corresponding control vector. D, E Caki-1 cells were transfected with an miR-629-5p mimic (D) or miR-629-5p inhibitor (E) for 24 h. NEDD4L protein levels and invasive ability of cells were respectively determined by Western blot (WB) (left panel) and Matrigel invasion (right panel) assays. F Expressions of KSRP and NEDD4L (left panel) and invasive ability of cells (right panel) were determined in Caki-1 cells transfected with pEGFP-KSRP with or without an miR-629-5p inhibitor. G Correlation between miR-629-5p and NEDD4L expression in TCGA KIRC patients as demonstrated in dot plots. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. H GSEA of TCGA KIRC patients with high (top 5%) versus low (bottom 5%) expressions of miR-629-5p. A gene set of the epithelial-mesenchymal transition (EMT) derived from HALLMARK was used. The normalized enrichment score (NES) and FDR are shown in the enrichment plot. I Correlations of miR-629-5p expression with EMT markers were demonstrated in a correlation plot using miRNA sequencing data of TCGA KIRC patients. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. J WB analysis of E-cadherin, N-cadherin, and vimentin expressions in Caki-1 and 786-O cells transfected with an miR-629-5p mimic. K Left panel, Schematic of the human NEDD4L-3' untranslated region (3'UTR) inserted in the luciferase reporter vector. Positions of AU-rich elements (AREs) are indicated by two gray oblongs, and the corresponding sequences are shown underneath. Right panel, Relative luciferase activities of Caki-1 cells transfected with the NEDD4L luciferase 3’UTR reporter vector containing wild-type or mutant ARE domains with or without KSRP shRNA. Values are presented as the mean ± SD of three independent experiments. **p < 0.01, compared to the respective control groups