Hepatic stellate cell-specific miR-214 expression alleviates liver fibrosis without boosting steatosis and inflammation

Abstract

Background: Liver fibrosis is a progressive pathological process primarily driven by the transdifferentiation of hepatic stellate cells (HSCs) into myofibroblast-like cells which secrete excessive extracellular matrix (ECM). Although microRNAs (miRNAs) have emerged as key regulators of fibrogenesis, the therapeutic potential and mechanistic specificity of miR-214-3p (miR-214) in liver fibrosis remain insufficiently defined.

Methods: An adeno-associated virus (AAV)-based system was used to achieve either whole-liver or HSC-specific overexpression of miR-214 (via GFAP promoter) in a mouse model of alcohol-associated liver fibrosis induced by Lieber-DeCarli ethanol diet combined with low-dose CCl₄ injection. Liver fibrosis, steatosis, and inflammation were evaluated by biochemical assays, histology, immunostaining, and gene expression analyses. In vitro, stable miR-214 overexpression and knockdown in LX-2 cells were performed to assess effects on HSC proliferation, transdifferentiation, and ECM gene expression. MECP2 was identified as a direct functional target of miR-214 by bioinformatics and luciferase reporter assays.

Results: miR-214 expression was significantly downregulated during HSC activation in vitro and in fibrotic livers. Whole-liver overexpression of miR-214 alleviated liver fibrosis but caused undesirable steatosis and inflammation. Notably, HSC-specific miR-214 overexpression ameliorated liver fibrosis without inducing these adverse effects. Functionally, miR-214 inhibited HSC proliferation and ECM gene expression, while its inhibition promoted this process. Mechanistically, miR-214 exerts its anti-fibrosis function at least in part by directing targeting MECP2, a critical regulator for HSC activation.

Conclusions: These findings not only identify miR-214 as a promising antifibrotic agent, but also highlight the translational advantage of cell-specific miRNA delivery. HSC-targeted miR-214 gene therapy may offer a promising and safer approach for treating liver fibrosis.

Keywords: AAV; Gene therapy; Hepatic stellate cell; Liver fibrosis; MiR-214.

Fig. 1

miR-214 is downregulated in activated HSCs and fibrotic liver. A Morphological images of primary HSCs during spontaneous activation from day 0 to day 2. B RT-qPCR analysis of miR-214 expression in primary HSCs during day 0 to day 2. C RT-qPCR analysis of miR-214 expression in LX2 cells treated with 3 ng/ml TGFβ for 6 h and 12 h. D RT-qPCR analysis of miR-214 expression in LX2 cells treated with 0.3 and 3 ng/ml TGFβ for 12 h. E Experimental design scheme of the alcohol-related fibrosis (ALF) model, which was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of low dose of CCl₄ (0.6 ml/kg·b.w.). F RT-qPCR analysis of miR-214 expression in the liver of Ctrl and ALF group. G, H Correlation analysis between miR-214 expression and the expression of Acta2 (G) and Fn1 (H). Ctrl, pair-treated group; ALF, alcohol-related fibrosis. **, p < 0.01

Fig. 2

Whole-liver miR-214 overexpression ameliorates liver fibrosis. A Schemic illustration of the experiment design. The fibrosis model was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of CCl₄. After 2 weeks, mice were administered with AAV8-miR-214 and AAV8-Ctrl via tail vain injection, and CCl₄ i.p. was hold for 1 week. B RT-qPCR analysis of the expression of miR-214 in the liver. C Serum ALT and AST levels. D Liver hydroxyproline levels. E Sirius Red staining and αSMA immunohistochemical staining of the liver tissues. F RT-qPCR analysis of the expression of Acta2 and Fn1 genes. n.s., non-significant; *, p < 0.05; **, p < 0.01

Fig. 3

Liver-wide miR-214 overexpression boosts liver steatosis and inflammation. A Liver weight. B Liver TG and TC levels. C H&E and Oil-Red O staining of the liver tissues. D RT-qPCR analysis of the gene expression related to fatty acid uptake and lipid metabolism. E F4/80 staining showing liver macrophage infiltration in the liver. F RT-qPCR analysis of the expression of inflammation-related genes. n.s., non-significant; *, p < 0.05; **, p < 0.01

Fig. 4

HSC-specific miR-214 overexpression alleviates liver fibrosis. A Experimental design scheme. The fibrosis model was established by combining Lieber-DeCarli liquid ethanol diet and i.p. injection of CCl₄. After 2 weeks, mice were administered with AAV8-GFAP-miR-214 and AAV8-GFAP-Ctrl via tail vain injection. B The expression level of miR-214 in the liver. C Serum ALT and AST levels. D The expression level of the fibrosis marker genes. E Sirius Red staining of liver tissues. F, G Western blotting showing the expression of proteins in the Smad signal pathway. n.s., non-significant; *, p < 0.05; **, p < 0.01

Fig. 5

HSC-specific miR-214 overexpression does not promote steatosis and inflammation. A Liver TC and TG levels. B Oil-Red O staining of liver tissues. C RT-qPCR analysis of the expression of genes related to lipid metabolism. D F4/80 staining showing liver macrophages. E RT-qPCR analysis of the expression of inflammation-related genes. n.s., non-significant; *, p < 0.05; **, p < 0.01

Fig. 6

miR-214 inhibits HSC proliferation. A Schematic illustrating the construction of miR-214 overexpression and inhibition plasmid. B RT-qPCR analysis of the expression of miR-214 in LX2 cells stably expressing (miR-214 OE) or inhibiting miR-214 (miR-214 TuD). miR-214 OE and miR-214 TuD lentivirus were packaged and LX2 cells were infected. Positive cells were selected by puromycin screening. C, D Cell proliferation ability of LX2 cells with miR-214 overexpression (C) or inhibition (D). E, F Colony images (E) and number/area analysis (F) of LX2 cells with miR-214-OE and miR-214 TuD. G, H Cell cycle analysis showing the cell cycle distribution of LX2 cells with miR-214 inhibition (G) or overexpression (H) group. **, p < 0.01

Fig. 7

miR-214 inhibits ECM gene expression. A KEGG pathway enrichment analysis of miR-214 using the mirPath v.3 tool. B RT-qPCR analysis of the expression of ECM-related genes. C Immunoblotting analysis of the expression of ECM-related proteins. *, p < 0.05; **, p < 0.01

Fig. 8

MECP2 is a potential target of miR-214. A Nuclear-cytoplasmic fractionation assay showing the cellular localization of miR-214 in LX2 cell. B Prediction of potential target genes of miR-214 using the TarBase, miRDB, TargetScan, and ENCORI online tools. C The expression levels of MECP2 in miR-214 overexpressing LX2 cells. D RT-qPCR analysis of the expression of Mecp2 in liver-wide and HSC-specific miR-214 overexpression group. E Cell ability analysis of MeCP2 overexpression in miR-214 OE LX2 cells. F Targeted binding site of miR-214 to MECP2 mRNA in human and mouse. G Luciferase reporter gene assay showing the direct binding of miR-214 to MECP2 mRNA. *, p < 0.05; **, p < 0.01