Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BackgroundIn chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.
MethodologyHSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.
Principal FindingsThe 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.
ConclusionsUpregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

研究背景：在慢性肝病中，肝星状细胞（hepatic stellate cells, HSC）会转分化为肌成纤维细胞，促进细胞外基质（extracellular matrix, ECM）的合成与沉积。转化生长因子-β（transforming growth factor-β, TGF-β）对肝星状细胞的刺激是肝纤维化发生的关键事件，因其可调控肌成纤维细胞转化并诱导细胞外基质生成。与之相反，肝细胞生长因子（hepatocyte growth factor, HGF）具有抗纤维化活性。近期有研究表明，miR-29参与细胞外基质的合成过程。因此，本研究探讨了HGF与TGF-β对肝星状细胞内miR-29胶原轴的调控作用。
研究方法：从大鼠体内分离得到肝星状细胞，在培养诱导其发生肌成纤维细胞转化的过程中，通过实时荧光定量PCR（Real-Time PCR）与蛋白质免疫印迹（Western blotting）技术，检测HGF及其受体Met（hepatocyte growth factor receptor, Met）的表达水平。随后，分别用HGF与TGF-β刺激肝星状细胞，或是通过胆管结扎构建大鼠实验性纤维化模型，检测此时TGF-β、HGF、Ⅰ型及Ⅳ型胶原的mRNA水平，同时检测miR-29a与miR-29b的表达量。通过报告基因实验分析miR-29与胶原mRNA 3′端非翻译区（3′-untranslated mRNA regions, UTR）的相互作用。采用miR-29模拟物（miR-29-mimicks）处理肝星状细胞，通过实时荧光定量PCR与免疫印迹实验，探究miR-29对胶原合成的抑制作用。
主要研究结果：本研究证实，Ⅰ型与Ⅳ型胶原的3′UTR可与miR-29结合。因此，在肝星状细胞中过表达miR-29a/b可显著降低Ⅰ型与Ⅳ型胶原的合成。反之，在体内实验性纤维化模型的胶原积累过程，以及体外TGF-β刺激肝星状细胞后，均观察到miR-29的表达水平下调。此外，研究发现，在肝星状细胞发生肌成纤维细胞转化以及TGF-β处理后，其肝细胞生长因子受体Met的表达会上调。综上，TGF-β刺激可降低miR-29的表达，解除其对胶原合成的抑制；而HGF刺激则可显著提升miR-29的表达水平，进而明显抑制Ⅰ型与Ⅳ型胶原的合成。
研究结论：HGF介导的miR-29上调与TGF-β介导的miR-29下调，分别参与了肝星状细胞的抗纤维化与促纤维化应答过程。