Fig. 8
CircPPFIA1 negatively regulates RAB36 expression by sequestering HuR. A Venn diagram representing overlapping genes by comparing RNA sequencing data with HuR CLIP-seq data. B Association of HuR with RAB36 mRNA was checked by HuR RIP. The enrichment of RAB36 mRNA in HuR RIP was determined by RT-qPCR. C The expression levels of RAB36 protein and mRNA were compared in KM12C and KM12L4 cells by western blot and RT-qPCR analyses, respectively. D The expression level of RAB36 in primary and liver metastatic colon cancer tissues was determined by RT-qPCR. E Western blot and RT-qPCR analyses were used to detect RAB36 protein and mRNA upon HuR silencing by two independent siRNAs. F The effect of HuR silencing on the stability of RAB36 mRNA was examined in KM12L4 treated with actinomycin D at the indicated time point. Half-lives of RAB36 mRNA were determined by calculating the time (t1/2) with 50% mRNA remaining. G–I The effect of circPPFIA1s silencing on RAB36 expression was examined by western blot analysis (G), HuR RIP (H), and the stability assay (I) using actinomycin as described above. J, K To examine whether overexpression of circPPFIA1s decreases RAB36 expression, KM12L4 cells were transfected with blank or overexpression vectors. The expression levels of RAB36 protein and mRNA were determined by western blot and RT-qPCR, respectively (J). The effect of circPPFIA1s overexpression on the interaction between HuR and RAB36 mRNA was examined by HuR RIP (K). L To examine whether knockdown of HuR reverses upregulation of RAB36 by knockdown of circPPFIA1s, western blot analysis was performed. M, N Following the transfection of KM12L4 cells with RAB36 siRNA, the efficacy of CDX1 siRNA was examined by western blot and RT-PCR (M). The effect of RAB36 silencing on the metastatic potential of KM12L4 cells was checked by transwell invasion and migration assays (N). Statistical analyses were performed using the Student’s t-test using three independent experiments (*p < 0.05). All data represent mean ± standard variation (SD)