TWIST2 is a direct functional target of miR-138. (A) The conserved miR-138 binding sequence of TWIST2 or its mutated form was inserted into the C-terminal of the luciferase gene to generate pGL3-TWIST2-WT or pGL3-TWIST2-MUT, respectively. (B) Ectopic expression of miR-138 down-regulated the TWIST2 mRNA level in HCT116 cells, as determined by quantitative real-time PCR. (C) miR-138 decreased the TWIST2 protein level in HCT116 cells, as determined by Western Blot. The TWIST2 protein band intensities were quantified and normalized to β-actin intensities. (D) miR-138 targets the wild-type but not the mutant 3′UTR of TWIST2. miR-138 was cotransfected with pGL3-Control(the empty vector) or reporters constructed containing either wild-type or mutant TWIST2 3′UTR into 293 T cells. pGL3-Control was used as an internal control reporter. The luciferase activity assay was repeated three times. **P < 0.01. (E) Levels of TWIST2 mRNA is up-regulated in primary colorectal tumors compared with their adjacent normal tissues (n = 36). (F) Correlation between TWIST2 protein expression and miR-138 expression in CRC tissues. P = 0.0039, R2 = 0.2608. n = 36 (G) Rescue assays using HCT116 cells co-transfected with Vector-TWIST2 or Vector and miR-138 or Control. **P < 0.01.