Figure 7

TGF-β1-mediated downregulation of miR-29 expression in PSCs is SMAD3 dependent.

(a) Predicted SMAD3 binding sites upstream of miR-29a/b1 promoter: there are two SMAD3 binding elements (S3BE) 5 kb and 49 kb upstream of the transcription start site of the miR-29a/b1 loci on chromosome 7. SMAD3 binds specifically to CAGA boxes: AG(C/A)CAGACA and regulates neighboring gene expression. (b) siSMAD3 efficiently reduces endogenous SMAD3 protein levels in hPSCs. hPSCs growing in culture were transfected with 50 nM non-targeting siRNA (siCTRL) or siSMAD3. 24 hours post-transfection, total proteins were harvested and subjected to western blot analysis to determine SMAD3 expression levels. GAPDH was used as a loading control. (c) SMAD3 knockdown abrogates TGF-β1-mediated miR-29 repression. qPCR analysis of miR-29 levels in TGF-β1-activated hPSCs transfected with siCTRL and siSMAD3. hPSCs were transfected with 50 nM siCTRL or siSMAD3. 24 hours post-transfection, cells were challenged with 10 ng/ml TGF-β1 for 24 hours and subjected to qPCR for miR-29 expression levels. All experiments were repeated three times and representative data is presented. Data are presented as mean + SEM; n = 3, statistics generated by t-test, *p < 0.05.