TGF-beta mediates transcriptional repression of miR–29 a/b1 during liver fibrosis

Background and aim: microRNA(miRNA) are small noncoding RNAs that play an important role in post-transcriptional inhibition of gene expression. Recent studies identified miR–29 as an antifibrotic miRNA, regulating collagen–1 and –4 expression in hepatic stellate cells (HSC). Additionally, the members of the miR–29 family were shown to be downregulated in livers from patients with advanced hepatic fibrosis. In the present approach, we studied the influence of the profibrotic mediator TGF-beta on promoter regulation of miR–29 expression.

Methods: The expression of pri-miRNA29a/b1 and mature miR–29a/b1 was determined by Real-Time PCR in HSC-T6 after TGF-beta stimulation. The promoter of the miR–29a/b1 was analysed by bioinformatics using the rVista 2.0 program and studied for transcription factor binding. Electrophoresis mobility shift assay (EMSA) and reporter assays were carried out to detect transcriptional miR–29 regulation.

Results: Quantification of pri-miR–29a/b1 and mature miR–29 levels in TGF-beta stimulated HSC clearly revealed that TGF-beta is a prominent regulator, decreasing miR–29 expression. Promoter analyses identified putative smad- and Ap–1 binding sites in the uptream regulatory domain of the miR–29 promoter. An Ap–1 binding site closed to the TATA-box was highly conserved between species. Reporter assays and EMSA pointed out that TGF-beta controlled miR–29 suppression by Ap–1 activation in HSC.

Conclusion: TGF-beta acts as a transcriptional repressor of miR–29 a/b1 expression and several AP–1 and smad binding sites were identified to direct miR–29 a/b1 promoter repression.

AP-1 - TGF-beta signaling - miR-29 - microRNA - promoter analyses - smad proteins