miR-133a mediates TGF-β-dependent derepression of collagen synthesis in hepatic stellate cells during liver fibrosis

doi:10.1016/j.jhep.2012.11.022

Journal of Hepatology

Volume 58, Issue 4, April 2013, Pages 736-742

https://doi.org/10.1016/j.jhep.2012.11.022 Get rights and content

Background & Aims

miRNAs are novel regulators of organ fibrosis. miR-133a plays a role in cardiac and muscle remodeling, but its function in the liver is unclear. We therefore aimed at evaluating a possible function of miR-133a in hepatofibrogenesis.

Methods

miR-133a levels were measured in whole liver samples from different murine hepatic fibrosis models and human liver tissue from patients with liver cirrhosis. The cell-specific regulation of miR-133a was assessed in FACS-sorted hepatic cell subpopulations. Murine and human primary hepatic stellate cells (HSC) were isolated and treated with different cytokines to evaluate upstream regulators of miR-133a. Moreover, GRX cells were transfected with synthetic miR-133a and the effect on extracellular matrix (ECM) gene regulation was assessed. Finally, miR-133a serum levels were measured in a cohort of patients with chronic liver diseases and correlated with disease progression.

Results

Overall miR-133a expression levels were unchanged in whole RNA extracts from fibrotic murine and human livers. However, miR-133a was specifically downregulated in HSC during fibrogenesis. Treatment of primary murine and human HSC with transforming growth factor (TGF)-β resulted in a significant downregulation of miR-133a in these cells. In turn, overexpression of miR-133a in primary murine HSC led to decreased expression of collagens. In addition, miR-133a serum levels were increased in patients with chronic liver disease and indicated the presence and progression of liver cirrhosis.

Conclusions

Evidence is presented for a novel antifibrotic functional role of miR-133a in hepatofibrogenesis. miR-133a may thus represent a target for diagnostic and therapeutic strategies in liver fibrosis.

Introduction

Liver fibrosis is characterized by an excessive accumulation of extracellular matrix (ECM) proteins in response to chronic hepatic injury [1]. Over the last years, hepatic stellate cells (HSC) have been commonly recognized as the principal cell type responsible for ECM protein formation during hepatic fibrogenesis, and the cytokine transforming growth factor (TGF)-β represents one key factor stimulating collagen and ECM-production in these cells [2].

miRNAs recently emerged as a class of small RNAs that regulate whole networks of genes during different biological processes [3]. In the context of liver diseases, previous studies revealed a role for miRNAs in acute liver injury, viral hepatitis or hepatocarcinogenesis [4]. Moreover, systematic array analyses on liver tissue from mice and humans indicated important functional roles of distinct miRNAs, such as the miR-29, in hepatic fibrogenesis [5], [6].

Despite its function in liver disease, it has become evident that miR-29 family members regulate remodeling processes also in other organs in response to chronic injury (e.g., [7], [8]). Therefore, in contrast to miRNAs with organ-specific functions such as miR-122 [9], it is likely that other miRNAs might exist that play a more comprehensive, organ-independent function in the mediation of fibrosis. In this context, a role for miR-133a, a member of the miR-1/miR-133 cluster of miRNAs, was recently proposed in the setting of vascular and cardiac remodeling [10], [11]. Moreover, it was shown that delivery of miR-1/miR-133 into skeletal muscle promoted muscle regeneration and prevented scar formation [12].

Based on these previous findings, in the present study we examined the role of miR-133a in murine and human liver fibrosis. We show that, despite unchanged miR-133a levels in whole liver miRNA extracts from mice and humans, miR-133a is specifically downregulated in HSC during fibrogenesis. This suppression is mediated by TGF-β, but not tumor necrosis factor (TNF), and promotes derepression of distinct collagen types in these cells. Moreover, we demonstrate that serum levels of miR-133a are elevated in patients with chronic liver disease and indicate the presence and progression of liver cirrhosis.

Section snippets

Isolation of murine cells

Primary hepatocytes and hepatic stellate cells (for stimulation and transfection experiments) and CD45⁺ cells were isolated from mice on C57Bl/6-background at the age of 40–55 weeks (allowing a more efficient cell extraction of HSC than from younger animals) and cultured as described previously [5], [13], [14]. For in vivo analysis of miR-133a expression in control and fibrotic livers, two groups of 5 mice each received either a carrier substance (oil) or CCl₄. HSC and macrophages were sorted on

Expression of miR-133a is unaltered in whole miRNA extracts from human and murine fibrotic livers

To assess the function of miR-133a in liver fibrosis, we first performed qPCR analysis on RNA extracts from different tissues to evaluate miR-133a expression in mice. While highest expression levels of miR-133a were found in muscle and heart followed by lung and brain, comparable expression patterns of miR-133a were detected in liver, spleen and kidney (Supplementary Fig. 1).

miR-133a expression was next measured in whole miRNA extracts from fibrotic livers of Balb/c mice treated for 6 weeks with

Discussion

In the current study, we provide evidence for a functional role of miR-133a in murine and human liver fibrosis. Based on our findings, we propose that miR-133a is part of a novel signaling pathway that mediates TGF-β-dependent derepression of extracellular matrix genes (e.g., Col1a1, Col5a3) in hepatic stellate cells during hepatic fibrogenesis (Supplementary Fig. 7). In addition, the specific regulation of miR-133a in HSC correlated with an elevation of miR-133a in the serum of patients with

Financial support

This work was supported by the German Research Foundation (SFB-TRR57/P06), a starting grant of the European Research Council within the FP7 (ERC-2007-Stg/208237-Luedde-Med3-Aachen), the German Cancer Aid (Deutsche Krebshilfe Grant 110043), the Interdisciplinary Centre for Clinical Research “BIOMAT”, the Ernst Jung Foundation Hamburg and grants from the START program of the medical faculty Aachen to T.L. and C.R.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

The authors express their gratitude to Michaela Roderburg-Goor and members of the Luedde’s lab for helpful discussions. Moreover, we would like to thank Sibille Sauer-Lehnen and Carmen Tag from the SFB-TRR57 for support in cell-isolation.

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^†: These authors contributed equally to this work.

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Research ArticlemiR-133a mediates TGF-β-dependent derepression of collagen synthesis in hepatic stellate cells during liver fibrosis

Background & Aims

Methods

Results

Conclusions

Introduction

Section snippets

Isolation of murine cells

Expression of miR-133a is unaltered in whole miRNA extracts from human and murine fibrotic livers

Discussion

Financial support

Conflict of interest

Acknowledgements

Cell

Biochim Biophys Acta

J Autoimmun

Exp Hematol

Liver fibrosis

J Clin Invest

Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques

Expert Rev Gastroenterol Hepatol

Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis

Hepatology

Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture

J Infect Dis

Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis

Proc Natl Acad Sci USA