Figure 2.

SOX9 is a direct target of MIR133A. A. Sequence alignment of wild-type (WT) and mutant (MT) MIR133A target site in the 3’-UTR of SOX9. A human SOX9 3’-UTR containing the wild-type and mutant MIR133A binding sequence was cloned downstream of the luciferase reporter gene. B, C. A luciferase reporter plasmid containing the WT or MT SOX9 3’-UTR was co-transfected into SW48 and Caco2 cells with pre-MIR1 as a negative control or pre-MIR133A. Luciferase activity was determined using the dual luciferase assay. Results are shown as relative firefly luciferase activity normalized to Renilla luciferase activity. D. qRT-PCR analysis of SOX9 expression in SW48, Caco2, and SW480 cells. E. MIR133A overexpression decreased SOX9 protein expression in HCT116, SW48, SW480, and Caco2 cell lines. The protein levels of SOX9 were determined by western blotting and densitometry by using Image J, where GAPDH was used as a loading control. Representative data from at least three independent experiments are shown. Each bar represents mean fold alternation above or below control (± SD). Differences were considered as statistically significant *P<0.05, **P<0.01 compared with control (ns = not significant).