Abstract
During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-β and its type 1 receptor (TGFBR1) mRNA expression, TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-β-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-β signaling.
Highlights
Hepatic stellate cells (HSCs) are one of the major players in fibrotic processes associated with wound healing in chronic liver injury
Overexpression of miR-25 had no significant effect on the expression of hepatic stellate cells (HSCs) quiescence (Peroxisome proliferator-activated receptor gamma (PPAR-γ) [PPARG], E-cadherin [CDH1]) or activation (vimentin [VIM], alpha smooth muscle actin [ACTA2], collagen 1a1 [COL1A1]) markers examined by qRT-PCR when compared to LX-2 cells transfected with control miR (Fig. 1C), nor on the proliferation rate of the cells, as assessed by growth rate measurements (Fig. 1D) and metabolic activity (MTT assay) (Fig. 1E)
To assess whether the downregulation of transforming growth factor beta receptor type 1 (TGF-βRI) has a functional impact on TGF-β-induced HSC activation, we stimulated LX-2 cells with increasing concentrations of recombinant TGF-β1 and measured the expression of the HSC activation marker ACTA2 and COL1A1 mRNA by qRT-PCR
Summary
Hepatic stellate cells (HSCs) are one of the major players in fibrotic processes associated with wound healing in chronic liver injury. HSCs engage in complex cross-talk with adjacent cells to promote fibrosis progression As part of their phenotypic morphogenesis during activation, fundamental changes in gene www.nature.com/scientificreports/. Several miRNAs, e.g. miR-122 and miR-21 have been shown to play a role in the development of liver fibrosis, regulating processes including tissue inflammation, cell apoptosis, as well as activation of HSCs19. We have recently demonstrated an increased abundance of miRNA-25-3p (miR-25) in serum of children with cystic fibrosis (CF) who have no liver disease involvement, compared to both CF children with liver disease and healthy individuals[21] This led us to propose that miR-25 may play a role in regulating the development of liver disease, via repression of pro-fibrogenic pathways in HSCs and other liver cells. We suggest that miR-25 expression is part of a negative feedback loop during liver fibrosis that dampens the responsiveness of HSCs to persistent fibrotic stimuli and mitigates excessive collagen secretion
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