Fig. 1

c-Myc promoted the transcription of miR-141 by directly binding its promoter. a Western blotting confirmed the expression of endogenous c-Myc in c-Myc knockdown stable 5-8F and HNE1 cells (shc-Myc) and control cells (shCtrl). b and c The levels of miR-141 and pri-miR-141 were assessed by qRT-PCR in c-Myc knockdown 5-8F and HNE1 cells. d Western blotting validated the overexpression of c-Myc in c-Myc-overexpressing HEK293 cells (c-Myc) and control cells (Vector). e The levels of miR-141 and pri-miR-141 were analyzed by qRT-PCR in c-Myc-overexpressing HEK293 cells. f and g The dual-luciferase reporter assays detected the relative miR-141 promoter activity in c-Myc knockdown 5-8F and HNE1 cells and c-Myc-overexpressing HEK293 cells, normalized to pRL-TK. h A schematic diagram showing the locations of predicted c-Myc-binding sites in the miR-141 promoter region on chromosome 12p13.31 (forward strand) and the amplified regions of PCR for ChIP assays. BS1-BS5: the 5 binding sites of c-Myc in the miR-141 promoter region predicted by the JASPAR Database. i ChIP-PCR assays using antibodies specific for Flag tags validated the c-Myc-binding sites in the miR-141 promoter. a and d β-actin served as an internal control. b, c and e GAPDH served as an internal control for pri-miR-141 detection, and U6 for miR-141 detection. The error bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001