Fig. 4

CARHSP1 directly regulates IL-17RA mRNA stability by binding to the 3’-UTR of IL-17RA. (A) KEGG pathway enrichment analysis of differentially expressed genes in 22Rv1 cells transfected with shCAR or shCon from the RNA-seq data. (B, C) Correlation analysis between mRNA expression levels of CARHSP1 and IL-17RA in PCa based on the TCGA cohort using TIMER (B) and GEPIA (C) database. (D, E) Effect of CARHSP1 knockdown on the expression of IL-17 receptor family members IL-17RA, IL-17RB and IL-17RC in 22Rv1 and PC-3 cells, as detected by RT-qPCR (n = 3, mean ± SD). 18 S was used as an internal loading control. (F) Western blotting analysis of the change of IL-17RA protein levels after CARHSP1 knockdown in 22Rv1 and PC-3 cells. β-actin was used as a loading control. (G) Cell surface analysis of IL-17RA protein using flow cytometry in 22Rv1 and PC-3 cells with stable depletion of CARHSP1 (n = 4, mean ± SD). (H) Measurement of IL-17RA mRNA stability by RT-qPCR after 22Rv1 and PC-3 cells transfected with shCAR or shCon were exposed to Actinomycin D (Act D) (5 μg/mL). The data are expressed as the percentage of mRNA molecules before the Actinomycin D treatment (n = 3, mean ± SD). (I) Diagram of the predicted CARHSP1 binding sites on the IL-17RA mRNA 3’ untranslated region (3’-UTR). (J) Analysis of the interaction of CARHSP1 with the IL-17RA 3’-UTR by RNA pull-down assays followed by western blot analysis in 22Rv1 and PC-3 cells. Vinculin was used as a loading control. NS RNA were random, non-specific binding RNAs that were used as negative controls. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant