Promoter regulation of the miR-29a/b1 gene by transforming growth factor-β in hepatic stellate cells and in hepatoma cells

Introduction: The members of the miR-29 family, targeting genes involved in apoptosis and gene methylation, are downregulated during hepatocarcinogenesis. Furthermore, in hepatic stellate cells (HSC) they inhibit extracellular matrix formation and are markedly reduced during fibrogenesis and after exposure to TGF-β. Since in both, liver fibrogenesis and carcinogenesis, TGF-β is a central mediator, we studied miR-29 promoter regulation by TGF-β in hepatic stellate and hepatoma cells as representative cell types for hepatic fibrosis and cancer.

Methods: Primary and mature miR-29 transcripts were quantified in HSC and hepatoma cells after TGF-β stimulation by Real-Time PCR. Transcriptional miR-29a/b promoter regulation was studied by reporter assays and electrophoresis mobility shift assays (EMSA). Putative binding sites were further characterized by site-specific mutation analyses and transgenic transcription factor activation.

Results: Quantification of cellular levels of primary transcripts of the miR-29a/b1 gene in addition to the mature processed forms revealed a pronounced upregulation in TGF-β stimulated hepatoma cells, but a downregulation in HSC. Reporter assays of truncated promoter constructs revealed that the core promoter, including a wide spectrum of putative transcription binding sites, is mainly involved in the TGF-β control. Whereas in HSC miR-29 repression by TGF-β stimulation is mainly affected by Smad binding sites, reporter assays of numerous site-specific mutation constructs as well as binding studies and supershift analyses, confirmed the central function of a Ap1/Ets-1 binding region in TGF-β mediated miR-29 induction in liver cancer cells.

Conclusion: miR-29a/b gene is controlled by a complex signaling network, in which Ets/Ap1 binding alteration is crucial for transcriptional induction in hepatoma cells.

Corresponding author: Odenthal, Margarete

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