Figure 1
ZEB1 was a negative transcription regulator of NT-induced miR-133α expression in human colonic NCM460-NTR1 cells.
(A) NT exposure (100 nM, 1 hr) reduced ZEB1 nuclear binding as evaluated in ChIP assay with nuclear extracts from NCM460-NTR1 cells incubated with NT and its vehicle control. (B) Expression of ZEB1 was reduced in NCM460-NTR1 cells transfected with si-ZEB1 as examined by RT-PCR when compared with cells transfected with scramble controls. (C) ZEB1 gene silencing in NCM460-NTR1 cells increased miR-133α levels at unstimulated condition as examined by RT-PCR when compared with cells transfected with scramble controls. (D) Increased miR-133α promoter-driven luciferase activities were detected from NCM460-NTR1 cells transiently transfected with si-ZEB1 when compared with cells transfected with scramble controls at unstimulated condition. (E) ZEB1 gene silencing reduced AFTPH levels were detected by RT-PCR in NCM460-NTR1 cells when compared with cells transfected to scramble controls. (F) Reduced AFTPH 3′UTR-driven luciferase activities were detected in ZEB1 gene-silenced NCM460-NTR1 cells when compared to cells transfected with scramble controls, as measured in luciferease assay. (G) No significant increase in luciferase activities was observed in cells transfected with plasmid expressing miR-133α promoter without ZEB1 binding site in the presence NT exposure (100 nM, 1 hr), when compared to those transfected with plasmid with ZEB1 binding site. *p < 0.05, **p < 0.01 when compared with control.
