MicroRNA as novel biomarker for disease severity evaluation and therapeutic target in NAFLD

초록

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is chronic and progressive liver disease with high prevalence of 30% of the general population. As global prevalence of NAFLD is increasing, it has become a major public health problem and major cause of chronic liver disease. Non-alcoholic steatohepatitis (NASH) with hepatic fibrosis has poor prognosis compared with non-alcoholic fatty liver (NAFL) or NASH without hepatic fibrosis. Micro RNA (miR) is a non-coding RNAwith about 20 nucleotides, and it can bind to the target mRNA, resulting in epigenetic reprogramming. Recently, many studies found that miRNA could be used as therapeutic targets and novel biomarkers for diagnosis and evaluation of severity in various diseases including NAFLD. Method: Circulating miRNAs were analysed in sera from 24 patients with biopsy-proven NAFLD using small RNA sequencing. We treated palmitic acid to various cell lines to induce lipotoxicity, and checked miRNA4449 expression in supernatant and cells. We identified merlin, an important inhibitory regulator of YAP-TAZ signaling, as the target of miR-4449 by TargetScan (targetscan.org). We confirmed in human liver tissue whether the expression level of merlin changes depending on the presence of fibrosis, and tested the interaction with miR-4449 in vivo. Results: Among 24 NAFLD patients, 15 patients were NAFL or NASH without fibrosis, whereas 9 patients were NASH with fibrosis. 31 miRNAs showed significant difference in expression level between two groups and miR-4449 was the most prominent among miRNAs that showed higher expression level in NASH with fibrosis compared to NAFL or NASH without fibrosis. Expression of miR-4449 increased in most supernatant and pellets from various cell lines when lipotoxicity was induced (Figure 1.). Therefore, hepatocytes are major source of miR-4449 during lipotoxicity and miR-4449 can be excreted to extracellular milieu. mRNA sequencing and quantitative PCR were performed on human liver tissue. Three patients were simple steatosis or NASH without fibrosis and three patients were NASH with fibrosis. Merlin showed significantly decreased in group of NASH with fibrosis (Figure 2.). When miR-4449 mimics and inhibitors were treated to Hep 3B cell lines, miR4449 mimic suppress merlin expression, whereas miR-4449 inhibitor increase expression of merlin (Figure 3.). Conclusion: miR-4449 might be used for novel therapeutic target of NASH-fibrosis.