Fig. 5

STAT3 activation downregulates miR-506-3p expression. (a) Overexpression of STAT3 didn’t cause an increase in FoxQ1 promoter luciferase activity in HCT116 cells. (b) Three independent miRNA target databases were used to predict the potential miRNAs. (c) Relative expression levels of representative nine potential miRNAs were detected in HCT116 transfected with STAT3 expression vector or empty vector as determined by RT-PCR. Error bars, SEM. (d) CRC cells (HCT116 and HT29) were infected with indicated siRNAs (si-STAT3) and treated with IL6 (50 ng/ml) or control for 48 h, and expression of miR-506-3p was examined using RT-PCR; Error bars, SEM. (e) Serially truncated and mutated miR-506-3p promoter constructs were cloned to pGL3-Basic luciferase reporters and transfected into HCT116 cells. The relative luciferase activities were determined after IL6 (50 ng/ml) treatment for 1 h; Error bars, SD. (f) Selective mutation analyses identified STAT3-responsive regions in the miR-506-3p promoter in HCT116 cells; Error bars, SD. (g) ChIP assay demonstrated the direct binding of STAT3 to the miR-506-3p promoter, including nonspecific control (N.C), CHIP1, and CHIP2 in HCT116 cells. Input, 5% of total lysate. (h) RT-PCR of the ChIP products confirmed the direct binding capacity of STAT3 to the miR-506-3p promoter in HCT116 cells. Input, 5% of total lysate; Error bars, SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001