Fig. 4
PUS10 inhibits RCC migration by promoting the maturation of miR-194-5p. A Schematic of our downstream candidate screening strategy. B Volcano plot of our microRNA sequencing results showing differentially expressed microRNAs in RCC tissues compared with adjacent normal tissues. (|log2FC| > 1 and FDR < 0.01) Twenty-nine downregulated (blue) microRNAs in RCC were identified. C Heatmap showing the downregulation of 29 microRNAs in our tissue. D Pearson correlation analysis demonstrated that the expression of miR-194-5p was positively correlated with that of PUS10 in 266 RCC tissues according to TCGA database. (r = 0.2913, P value < 0.0001). E Kaplan‒Meier analysis showed the correlation between the expression of miR-194-5p and the overall survival of patients based on the TCGA cohort. F The decreased expression of miR-194-5p in RCC was verified in SRRSH patient-derived tissues by performing qRT‒PCR. G A positive correlation between the expression of miR-194-5p and PUS10 was identified in the SRRSH cohort. (r = 0.44, P value < 0.05). H qRT‒PCR assay showed that the expression of miR-194-5p and pre-miR-194 were repressed after the transfection of siRNA targeting PUS10 in 786-O and Caki-1 cells, while their primary precursor pri-miR-194 accumulated. I qRT‒PCR assay showed that the expression of miR-194-5p and pre-miR-194 were increased after the ectopic expression of PUS10 in RCC cells, with their precursor pri-miR-194 being further consumed. J The interaction between PUS10 and pri-miR-194 was determined by performing an RNA immunoprecipitation (RIP) assay and qRT‒PCR with an anti-Flag antibody in Caki-1 and 293T cells transfected with a flag-tagged PUS10 plasmid. K Immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) assays exhibited the co-localization of PUS10 and pri-miR-194 in the nucleus of RCC cells. L RIP assays showed that the enrichment of pri-miR-194 on the microprocessor protein DGCR8 was impaired by the depletion of PUS10 in Caki-1 cells. M Compensation of miR-194-5p in Caki-1 and 786-O cells abolished the enhanced migration induced by PUS10 knockdown. Migrating cells in three replicate experiments were counted and are presented as the mean ± SD in the histogram. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant