MIR-4449 REGULATES NASH-FIBROSIS PROGRESSION BY MODULATING THE MERLIN AND TAZ PATHWAYS

Abstract

Background: Although most non-alcoholic fatty liver disease (NAFLD) patients show benign clinical courses, non-alcoholic steatohepatitis (NASH) patients with hepatic fibrosis have poor prognoses compared to patients with non-alcoholic fatty liver (NAFL) or NASH without hepatic fibrosis. In this study, we aimed to analyze the role of miR-4449 in the progression of NASH-fibrosis.

Methods: Liver tissue and sera were collected from NAFLD patients who received liver biopsies in Korea University Guro hospital. MicroRNA sequencing, using sera, and mRNA sequencing, using liver tissue, were performed in patients with biopsy-confirmed NAFLD. To induce in vitro lipotoxicity in mice hepatocytes, HepG2 and Huh7 cells were treated with palmitic acids (PA). We transfected miR-4449 mimic or inhibitor into hepatocytes to explore the effect of miR-4449 during lipotoxicity. Mice were fed either a normal diet, a high fat diet, or high fat with fructose for 24 weeks. The high fat with fructose group were also treated with an miR-4449 mimic or miR-4449 inhibitor administered through tail veins.

Results: In total, 24 NAFLD patients were recruited, 15 patients had NAFL or NASH without fibrosis, whereas nine patients had NASH with fibrosis. In miRNA sequencing analysis, 31 miRNA sequences showed significant differences in expression levels between the two groups, with the expression of miR-4449 most prominently seen among miRNAs that showed higher expression levels in NASH-fibrosis compared to the NAFL or NASH without fibrosis group. PA treatment increased the expression level of miR-4449 in both supernatant and hepatocytes. On the other hand, the expression of merlin, which could be the target of miR-4449 , decreased in PA-treated hepatocytes compared with vehicle-treated hepatocytes. In addition, merlin expression levels significantly decreased in NASH patients with fibrosis compared to NAFL and NASH patients without fibrosis. Hepatocytes with miR-4449 transfection mimic decreased merlin expression but exhibit increased phosphorylated TAZ expression, whereas hepatocytes transfected with miR-4449 inhibitor demonstrate increased merlin expression but decreased phosphorylated TAZ expression. The high fat with fructose group increased expression levels of miR-4449 in the liver compared with the high fat group. Treatment with miR-4449 inhibitors ameliorated liver steatosis and fibrosis, whereas treatment with miR-4449 mimics aggravated liver steatosis and fibrosis.

Conclusion: Patients with NASH-fibrosis showed increased miR-4449 expression. miR-4449 regulates merlin expression and TAZ phosphorylation in hepatocytes during lipotoxicity. miR-4449 may be of benefit as a novel therapeutic target in NASH-fibrosis.