Figure 4: miR-193a suppresses the progression of CT26 colon cancer by directly targeting Caprin1.

(a) Schematic diagram of the putative binding sites of miR-193a in the wild-type (WT) Caprin1 3′ untranslated regions (UTR). The miR-193a seed matches in the Caprin1 3′UTR are mutated at the positions as indicated. CDS, coding sequence. (b) Potential miR-193a binding sites on Caprin1 (in grey) are broadly conserved among vertebrates. (c) Expression of miR-193a and candidate target gene Caprin1 as well as downstream genes (Ccnd2, c-myc) in CT26 cells assessed by qPCR following transfection of miR-193a mimic and control scramble miRNA. (d) Expression of candidate miR-193a and candidate target genes Caprin1 as well as downstream genes (Ccnd2, c-Myc, G3bp1) in CT26 cells assessed by western blot, following transfection of miR-193a mimic and control miRNA for 72 h. (e) Proliferation of CT26 cells with miR-193a and potential target Caprin1 knock down. Cell viability was detected from day 0 to 5 after transfection. (f) Luciferase activity assays of wild-type (WT) and mutated Caprin1 3′UTR luciferase reporters after co-transfection with miR-193a mimic, miRNA mimic control (scramble), anti-sense miR-193a or anti-sense negative control RNA in CT26 cells. The luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of transfected control mimic miRNA was set as a relative luciferase activity of 1. (g) Survival of BALB/c mice after intrasplenic injection of CT26 cells with miR-193a overexpression (n=6 mice per group). (h) The cell cycle phase analysis of CT26 cells transfected with miR-193a mimic for 72 h using PI. The percentage of cells in the G1, S, and G2 phases are shown in the bar graph. (i) BrdU incorporation assay for cell cycle analysis of CT26 transfected with miR-193a mimic and control miRNA. Error bars represent s.e.m. *P<0.05 and **P<0.01 (two-tailed t-test). Each data point was measured in triplicate (error bars, s.e.m.).