CXCR4 is a direct target of miR-133b. (A) Schematic of the luciferase reporter assay used to validate the interaction between miR-133b and the 3′UTR of CXCR4. Grey font indicates the ‘seed’ regions. The MRE CXCR4 3′UTR of wild, full mutant and full length were separately inserted into a psiCHECK-2 vector downstream from the Renilla luciferase gene. (B-C) Dual luciferase reporter assays performed using the 59 nt flanking the MRE, full mutation in the MRE (B) and the entire 3′UTR of CXCR4 (C). (D) miR-133b expression was measured using qRT-PCR in SW-480 and SW-620 human CRC cell lines and normalized to U6 snRNA expression. (E) miR-133b suppressed CXCR4 expression in CRC cells. CXCR4 protein expression of CRC cells transfected with miR-133b mimic, miR-133 inhibitor, siCXCR4 or a negative control normalized to GAPDH expression. Data are shown as the mean ± SD from three independent assays. *P < 0.05, ** P < 0.01 compared to controls.