Figure 4

IGF-1R is experimentally validated as a co-target of miR-99b-5p and miR-203a-3p in GC cells. (A) Putative miR-99b-5p/203a-3p-binding sites in the IGR-1R 3′UTRs, mutations were generated in the IGF-1R 3′UTR sequences by mutating 4 nt for the seed region of miR-99b-5p/203a-3p, as indicated. (B) Dual luciferase assays were performed in HEK293 cells after co-transfection with the wild-type or mutant IGR-1R 3′-UTR plasmids and pre-miR-99b/203a. (C) The TCGA data of IGF1R mRNA expression in GC tissues (n = 35) and normal tissues (n = 435). Overall survival analysis showed that there was no statistically significant between IGF1R high expression and low expression tumors. (D) IGF-1R was determined by qRT-PCR in GC tissues (left). The correlation between miR-99b-5p/203a-3p and IGR-1R was analyzed. IGF-1R was determined by qRT-PCR and western blot in GC cell lines (right). β-actin was employed as a housekeeping control. (E,F) IGF-1R expression level was measured by qRT-PCR and western blot after transfection with pre-miR-99b/203a and anti-miR-99b-5p/203a-3p in MKN-45/SGC-7901 cells (*P < 0.05, **P < 0.01, Student’s t test or Mann-Whitney test).