Fig. 4: c-Met, Sox9, and WAVE2 are direct targets of miR-133b and are negatively correlated with miR-133b expression in breast cancer.

a Dual-luciferase assays showing that repression of potential target genes by miR-133b was measured by luciferase activity in MDA-MB-231 cells. The data are mean ± s. e. m. of separate transfections (n = 3), and are shown as the ratios of Renilla activity to Firefly activity. b A schematic of cloning miR-133b binding sites mutant 3′-UTR of target genes into the dual-luciferase vector. The substitutions of nucleotides within binding sites are shown in red. c Relative activity of the luciferase gene fused with the wild-type or mutant 3′-UTR of WAVE2, Sox9, and c-Met genes, respectively. The data are mean ± s. e. m. of separate transfections (n = 3), and are shown as the ratio of Renilla activity to Firefly activity. d Western blot analysis of c-Met, Sox9, and WAVE2 expression in multi-type breast cancer cell lines with or without miR-133b overexpression. Left two panels: Endogenous WAVE2, Sox9, and c-Met proteins were detected when MDA-MB-231 and BT549 were transfected with NC or miR-133b mimics for 48 and 72 h. Right panel: Immunoblotting of candidate proteins in MDA-MB-231-luc-miR-133b and control cells (plvx-vector). β-Actin serves as internal control. e Expression of c-Met, Sox9, and WAVE2 in six paired clinical breast cancer specimens. N and T mean adjacent normal tissue and paired breast carcinoma, respectively. Vinculin serves as internal control. f Correlation of miR-133b and target genes in paired clinical breast cancer samples. The expression level of WAVE2/Sox9/c-Met was designated as the ratio of optical intensity between target gene and vinculin in each line, whereas the relative abundance of miR-133b was determined by stem-loop RT-PCR. For miR-133b and WAVE2/Sox9/c-Met, their expression level was assumed as 1 in adjacent normal tissues and corresponding calculated in paired tumor tissues. The symbol * denotes statistical difference (p < 0.05), whereas ** and *** represent great significant difference (p < 0.01 and p < 0.001) by a two-tailed Student’s t-test