Transcriptional promoter regulation of the miR-29a/b1 gene in hepatoma cells

Background and aim: Recent studies have shown that members of the microRNA-29 (miR-29) family are dysregulated in several types of cancer. Dysregulation of miR-29 family members during hepatocarcinogenesis leads to a more malignant phenotype of hepatocellular carcinoma (HCC). Many potential targets of the miR-29 family were identified such as Mcl-1 and BCl2. In the present approach, we have studied promoter gene regulation of miR-29 expression.

Methods: The expression of pri-miRNA29a/b1 was determined by Real-Time PCR in Huh7 after TGF-beta or phorbolester stimulation. The promoter of the miR-29a/b1 was analysed by bioinformatics using the rVista 2.0 software. For investigation of transcription factor binding, nuclear extracts from Huh7 hepatoma cells were prepared and electrophoresis mobility shift assays (EMSA) were carried out. Transcriptional miR-29a/b promoter regulation was studied by reporter assays and putative binding sites were analysed after site-specific mutation and recombinant expression of the mutated promoter constructs in Huh7 cells.

Results: Quantification of cellular levels of primary transcripts of the miR-29a/b1 gene in addition to the mature processed forms of the miR-29a and miR-29b revealed a pronounced upregulation in TGF-beta or phorbolester stimulated Huh7. Promoter analyses identify putative Ap-1 binding sites in the upstream regulatory domain of the miR-29 promoter. An Ap-1 binding site close to the TATA-box was highly conserved between species. Reporter assays and EMSA pointed out that TGF-beta and phorbolester controlled pri-miR-29 expression through Ap-1 activation in hepatoma cells. Site-specific mutation constructs of several putative binding sites and subsequent reporter assays, binding studies and supershift analyses using pan-fos antibodies confirmed the central function of the transcription factor Ap-1 in miR-29 regulation.

Conclusion: miR-29a/b gene is controlled by a complex signaling network, in which the transcription factor Ap1 acts as a central transcriptional inducer of miR-29 a/b1 expression.