Fig. 3. FZD6 is a direct target of miR-199a-5p in CRC cells. (A) The putative binding sequence of miR-199a-5p in FZD6 3’-UTR is well conserved in various species (upper panel). Diagram shows the miR-199a-5p and a putative binding sequence in 3’-UTR of FZD6 mRNA predicted by Target scan algorithm (bottom panel). (B) qRT-PCR showed that endogenous expression of FZD6 mRNA in DLD1 cells was significantly downregulated by overexpression of miR-199a-5p. Data were normalized against GAPDH mRNA expression. Results are the average of three independent experiments conducted in duplicate. **P ＜ 0.01 (C) Western blot also revealed that overexpression of miR-199a-5p resulted in reduced expression of FZD6 at protein level. (D) Luciferase activity was significantly decreased in miR-199a-5p overexpressed DLD1 cells. Results are the average of three independent experiments conducted in duplicate. ***P ＜ 0.001.