Fig. 6

miR-506-3p downregulated FoxQ1 expression by directly binding its 3′UTR, and miR-506-3p/FoxQ1 is essential for STAT3-mediated CRC cell growth, migration, and invasion. (a) Western blot for FoxQ1 in six human CRC cell lines normalized as to expression of GAPDH. (b) RT-PCR results of miR-506-3p in indicated CRC cell lines normalized as to expression of U6; Error bars, SD. (c) Negative correlation between levels of FoxQ1 and miR-506-3p in CRC cell lines(r = 0.82). (d) Schematic representation of the FoxQ1 3′UTR. Mutations were generated at the predicted miR-506-3p–binding sites. (e) Luciferase assays demonstrated that expression of FoxQ1 3′UTR (WT or mutant form) by HEK293T cells transfected with miR-506-3p mimics or with control mimics. Error bars, SD. (f) Luciferase assays demonstrated that expression of FoxQ1 3′UTR (WT or mutant forms) by HEK293T cells transfected with miR-506-3p inhibitor or with control inhibitor. Error bars, SD. (G) CRC cells (HCT116 and HT29) were transfected with miR-506-3p mimics or inhibitor at a final concentration of 100 and 200 nmol/L, respectively. The levels of FoxQ1 mRNA were analyzed by RT-PCR at 48 h post transfection; Error bars, SEM. (h) Levels of FoxQ1 protein were analyzed by Western blot at 72 h post transfection. (i) The levels of E-cadherin and Vimentin were analyzed by Western blot at 48 h post miR-506-3p mimics transfection with or without TAMs co-culture. (j) HCT116 cells transfected with miR-506-3p mimics alone or in combination with FoxQ1 and their proliferation were analyzed by colony formation assays. Error bars, SD. (k) HCT116 cells transfected with miR-506-3p mimics alone or in combination with FoxQ1 were subjected to transwell migration and invasion assays. (magnification, × 200). Error bars, SD. (l) HCT116 cells transfected with STAT3 siRNA or with control siRNA alone or in combination with miR-506-3p inhibitor or FoxQ1 were subjected to colony formation assays. Error bars, SD. (m) HCT116 cells transfected with STAT3 siRNA or with control siRNA alone or in combination with miR-506-3p inhibitor or FoxQ1 were subjected to transwell migration and invasion assays. (magnification, × 200). Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001