Fig. 6
CircPCNXL2 upregulates SRSF1 expression by sponging miR-766-3p. (a) Venn diagram showed the overlap of the target miRNAs of circPCNXL2 predicted by Targetscan, miRanda and RNAhybrid. (b) The enrichment of 4 putative miRNAs was evaluated in the RNA pull down by circPCNXL2 probe in HuCCT1 cells. (c) The enrichment of circPCNXL2 and miR-766-3p was confirmed by RIP with AGO2 or IgG antibody in HuCCT1 cells. (d) The expression of miR-766-3p in 76 paired ICC tissues was measured by qRT-PCR. (e) Kaplan–Meier analysis of miR-766-3p expression and overall survival in 76 patients with ICC. The cutoff is the median expression value of miR-766-3p. (f) Correlation analysis showed a negative relationship between the levels of circPCNXL2 and miR-766-3p. (g) The wild-type (WT) and mutant (Mut) circPCNXL2 plasmid for dual-luciferase report were applied to confirm the interaction between circPCNXL2 and miR-766-3p. (h) The functions of miR-766-3p on the proliferation of HuCCT1 cells were detected by colony formation assay. (i) The functions of miR-766-3p on the migration of HuCCT1 cells was detected by transwell assay, scale bar = 100 μm. (j-k) The mRNA and protein levels of SRSF1 were evaluated in HuCCT1 transfected with miR-766-3p mimics. (l) The expression of SRSF1 in 76 paired ICC tissues was measured by qRT-PCR. (m) Kaplan–Meier analysis of SRSF1 expression and overall survival in 76 patients with ICC. The cutoff is the median expression value of SRSF1. (n-o) Correlation analysis showed a negative relationship between the levels of SRSF1 and miR-766-3p and a positive relationship between the levels of SRSF1 and circPCNXL2. (p) The wild-type (WT) and mutant (Mut) SRSF1 plasmid for dual-luciferase report were applied to confirm the interaction between miR-766-3p and SRSF1. (q) The functions of SRSF1 on the proliferation of HuCCT1 cells was detected by colony formation assay. (r) The functions of SRSF1 on the migration of HuCCT1 cells was detected by transwell assay, scale bar = 100 μm. (s-t) The levels of SRSF1, p-ERK and ERK were measured in HuCCT1 or RBE cells co-transfected with circPCNXL2 overexpression and/or SRSF1 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001