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Regulation of mesenchymal phenotypes by microRNA‐21 during alcohol‐induced liver injury
Author(s) -
Meng Fanyin,
Francis Heather,
McDaniel Kelly,
Alpini Gianfranco
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.516.12
Subject(s) - hepatic stellate cell , mesenchymal stem cell , liver injury , biology , timp1 , microrna , vimentin , andrology , microbiology and biotechnology , pathology , cancer research , medicine , endocrinology , immunology , gene expression , immunohistochemistry , gene , biochemistry
Background MicroRNAs belong to the heterogeneous class of non‐coding RNAs (ncRNAs) that regulate gene expression in a sequence specific manner. The activation of miR‐21 and mesenchymal signaling is recognized as the key event of alcoholic liver injury and hepatic fibrosis. The current study was aimed to characterize the functional role of microRNA‐21 regulated mesenchymal phenotypes in mice liver with chronic and binge ethanol feeding (the NIAAA model ) in vivo and activated human hepatic stellate cells (HSCs) in vitro . Methods miRNA expression in TGF‐β treated HSCs, as well as in 5 weeks chronic and binge ethanol feeding mouse liver specimen (with or without anti‐miR‐21 Vivo‐Morpholino treatment) and control HSCs and liver tissue, was assessed using a hybridization based microarray and by real‐time PCR, respectively. The mesenchymal phenotypes and the activation of HSCs were monitored by Western blot and real‐time PCR analysis through specific markers such as vimentin, S100A4, α‐SMA, laminin, fibronectin and MMPs. Results The total liver histopathology score increased in chronic ethanol feeding group relative to control mice, whereas Sirius red staining revealed increased liver fibrosis with hepatic stellate activation after 5 weeks ethanol feeding. We identified that ethanol feeding significantly up‐regulated miR‐21 by 5.3±0.8 fold, which was further verified by Taqman real‐time PCR assay. The expression of miR‐21 was also significantly increased during human hepatic stellate cell activation when cultured in uncoated plastic culture dishes for 5 weeks. Treatment of HSCs with TGF‐β (10 ng/ml) for 24 hours induced a significant increase of miR‐21 expression in both activated and control state. Transfection of anti‐miR‐21 inhibitor markedly attenuated the expression of S100A4, laminin and fibronectin mRNAs and additionally blunted the increased expression of α‐SMA, MMP‐2 and MMP‐9, which are key genes involved in the activation of HSCs. Furthermore, the expressions of TGF‐β, as well as mesenchymal and fibrosis markers, including vimentin, S100A4, α‐SMA, laminin, fibronectin, MMP‐2, MMP‐9 and LIN28, were significantly reduced after miR‐21 Morpholino/AS treatment in chronic ethanol feeding mouse liver specimens compared to controls. Conclusion Our results show that miRNAs are critical regulators of mesenchymal transition, human hepatic stellate activation and transdifferentiation during alcoholic liver injury. The findings provide new insight into the function of specific miRNAs and the mechanisms of alcohol induced liver injury and fibrosis.