Independent of its trigger the development of liver fibrosis is caused by the activation of hepatic stellate cells (HSCs). This transdifferentiation process can be directed by microRNAs (miR), small non-coding RNAs suppressing mRNA expression. Recently, we have shown that cystic fibrosis (CF) patients with accompanied liver disease reveal significantly lower serum levels of miR-CF+ compared to patients without liver disease, suggesting a potential protective antifibrotic effect of the molecule. The role of miR-CF in HSC activation has not been described yet. In this study, we identified and validated potential miR-CF targets in activated HSCs and examined the impact on HSC phenotype.
Materials and Methods
The human HSC cell line (LX-2) was transfected with biotinylated miR-CF duplexes, miR-CF mimics or control miRNA, respectively. After 24, 48 or 72h cells were lysed for subsequent experiments. Identification of potential target mRNA was performed by pull down with streptavidin magnetic beads, followed by Illumina iScan microarray analysis. Afterwards, potential targets were validated via luciferase reporter assay as well as qPCR and Western Blot expression analysis. To investigate the biological function of miR-CF in HSC activation, LX-2 were transfected with miR-CF and subsequently stimulated with TGFβ, a potent HSC activator, after 24h in culture. Expression of HSC activation markers was analysed in cell lysates using qRT-PCR.
Results and Conclusion
We identified 541 differentially expressed genes, some of which are directly linked with HSC activation (via PPARg or notch signalling). Indeed, (co-)activators of notch FKBP14 and ADAM-17, as well as activation-associated marker (MCP-1, Wnt5a) were downregulated in miR-CF treated HSC. Moreover, TGFβ-induced upregulation of collagen1a1 was decreased after miR-CF transfection. Hence, we suggest that miR-CF has a potentially antifibrotic effect by suppressing HSC activation capacity and may therefore be an attractive target for liver fibrosis therapy.
+ miRNA name de-identified due to IP protection