RNA-seq performed on the vehicle- and dexamethasone-treated ccRCC cells to identify putative NR3C1 downstream targets
We investigated the effects of modulating NR3C1 activity as well as targeting NR3C1 using CRISPR/Cas9 in human ccRCC in-vitro model. We found the activation of NR3C1 by dexamethasone enhanced ccRCC cells growth capability. In contrast, NR3C1 inhibition, either using mifepristone or CRISPR/Cas9, reduced ccRCC cells growth and spheroid-forming capabilities. RNA-Seq was performed on the vehicle- and dexamethasone-treated ccRCC cells to identify putative NR3C1 downstream targets that might be involved in driving the observed phenotypes. Out of the 475 differentially expressed genes, we chose and successfully validated two upregulated (PDK4 and EDN2) and two downregulated (IL-6 and CD274) genes. Moreover, dexamethasone treatment of the CRISPR/Cas9 NR3C1-targeted ccRCC cells neither upregulated nor downregulated of the respective chosen genes, suggesting NR3C1 role as the direct transcriptional activator of these genes. This was confirmed by the analysis of public NR3C1 ChIP-Seq and our previous H3K27ac ChIP-Seq data that showed NR3C1 binding at the PDK4, EDN2 and IL-6 upstream regulatory elements
We investigated the effects of modulating NR3C1 activity as well as targeting NR3C1 using CRISPR/Cas9 in human ccRCC in-vitro model. We found the activation of NR3C1 by dexamethasone enhanced ccRCC cells growth capability. In contrast, NR3C1 inhibition, either using mifepristone or CRISPR/Cas9, reduced ccRCC cells growth and spheroid-forming capabilities. RNA-Seq was performed on the vehicle- and dexamethasone-treated ccRCC cells to identify putative NR3C1 downstream targets that might be involved in driving the observed phenotypes. Out of the 475 differentially expressed genes, we chose and successfully validated two upregulated (PDK4 and EDN2) and two downregulated (IL-6 and CD274) genes. Moreover, dexamethasone treatment of the CRISPR/Cas9 NR3C1-targeted ccRCC cells neither upregulated nor downregulated of the respective chosen genes, suggesting NR3C1 role as the direct transcriptional activator of these genes. This was confirmed by the analysis of public NR3C1 ChIP-Seq and our previous H3K27ac ChIP-Seq data that showed NR3C1 binding at the PDK4, EDN2 and IL-6 upstream regulatory elements
universiti kebangsaan malaysia