Background Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through HSC activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. Methods Expression of pri-miR17-92 and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b and 92 were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from tissue culture media and plasma of rodent models and patients with ALD. miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. Results Pro-fibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD were similar to those seen in a rodent model of alcoholic injury and in vitro. Conclusions Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights to the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

Fibrotic liver injury is a significant healthcare burden in the United States. It represents a major cause of morbidity and mortality for which there are no effective Food and Drug Administration-approved treatment strategies. Fibrosis is considered a disruption of the normal wound healing responses mediated by fibroblastic cells, which are triggered and sustained by pro-fibrotic cytokines such as transforming growth factor beta 1 (TGF-β1). TGF-β1-mediated trans-differentiation of hepatic stellate cells (HSCs) from quiescent to activated myofibroblasts is a pivotal event in the development of fibrosis. Activation is accompanied by global changes in microRNA (miR) expression. It has been previously reported that miR19b is decreased in activated HSCs and contributes to increased expression of TGF-β receptor II and connective tissue growth factor, both confirmed targets of miR19b. An adeno-associated virus serotype 2 vector (AAV2) with a miR19b transgene downstream of enhanced green fluorescent protein under the murine collage alpha 1(I) promoter was developed specifically to target HSCs. Male Sprague Dawley rats (250 g) underwent sham or bile-duct ligation (BDL) surgery. Directly after BDL, rats received AAV2-miR19b, AAV2-control, or vehicle normal saline (NS) by portal-vein injection. After 2 weeks, the animals were euthanized, and blood was collected for alanine and aspartate aminotransferase, total and direct bilirubin, and alkaline phosphatase. Tissue was collected for RNA and protein extraction and histology. Fibrosis and measures of hepatic injury were significantly reduced in AAV2-miR19b-treated rats in combination with significant improvements in total and direct bilirubin. Histological analysis of collagen by PicroSirius Red staining revealed a ∼50% reduction compared to AAV2-control or NS-injected animals. Pro-fibrotic markers, smooth-muscle alpha-actin, TGF-β receptor II, and collagen alpha 2(I) mRNA and protein were significantly decreased compared to AAV2-control and NS groups. AAV2-mediated reintroduction of miR-19b, specifically expressed in HSCs, improved liver function, inhibited fibrosis, and improved measures of hepatic injury in a BDL model.

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