Differentially expressed mRNAs and lncRNAs shared between activated human hepatic stellate cells and nash fibrosis.

We previously reported dysregulated expression of liver-derived messenger RNA (mRNA) and long noncoding RNA (lncRNA) in patients with advanced fibrosis resulting from nonalcoholic fatty liver disease (NAFLD). Here we sought to identify changes in mRNA and lncRNA levels associated with activation of hepatic stellate cells (HSCs), the predominant source of extracellular matrix production in the liver and key to NAFLD-related fibrogenesis. We performed expression profiling of mRNA and lncRNA from LX-2 cells, an immortalized human HSC cell line, treated to induce phenotypes resembling quiescent and myofibroblastic states. We identified 1964 mRNAs (1377 upregulated and 587 downregulated) and 1460 lncRNAs (665 upregulated and 795 downregulated) showing statistically significant evidence (FDR ≤0.05) for differential expression (fold change ≥|2|) between quiescent and activated states. Pathway analysis of differentially expressed genes showed enrichment for hepatic fibrosis (FDR = 1.35E-16), osteoarthritis (FDR = 1.47E-14), and axonal guidance signaling (FDR = 1.09E-09). We observed 127 lncRNAs/nearby mRNA pairs showing differential expression, the majority of which were dysregulated in the same direction. A comparison of differentially expressed transcripts in LX-2 cells with RNA-sequencing results from NAFLD patients with or without liver fibrosis revealed 1047 mRNAs and 91 lncRNAs shared between the two datasets, suggesting that some of the expression changes occurring during HSC activation can be observed in biopsied human tissue. These results identify lncRNA and mRNA expression patterns associated with activated human HSCs that appear to recapitulate human NAFLD fibrosis.

AEBP1 expression increases with severity of fibrosis in NASH and is regulated by glucose, palmitate, and miR-372-3p.

Factors governing the development of liver fibrosis in nonalcoholic steatohepatitis (NASH) are only partially understood. We recently identified adipocyte enhancer binding protein 1 (AEBP1) as a member of a core set of dysregulated fibrosis-specific genes in human NASH. Here we sought to investigate the relationship between AEBP1 and hepatic fibrosis. We confirmed that hepatic AEBP1 expression is elevated in fibrosis compared to lobular inflammation, steatosis, and normal liver, and increases with worsening fibrosis in NASH patients. AEBP1 expression was upregulated 5.8-fold in activated hepatic stellate cells and downregulated during chemical and contact induction of biological quiescence. In LX-2 and HepG2 cells treated with high glucose (25 mM), AEBP1 expression increased over 7-fold compared to normal glucose conditions. In response to treatment with either fructose or palmitate, AEBP1 expression in primary human hepatocytes increased 2.4-fold or 9.6-fold, but was upregulated 55.8-fold in the presence of fructose and palmitate together. AEBP1 knockdown resulted in decreased expression of nine genes previously identified to be part of a predicted AEBP1-associated NASH co-regulatory network and confirmed to be upregulated in fibrotic tissue. We identified binding sites for two miRNAs known to be downregulated in NASH fibrosis, miR-372-3p and miR-373-3p in the AEBP1 3' untranslated region. Both miRNAs functionally interacted with AEBP1 to regulate its expression. These findings indicate a novel AEBP1-mediated pathway in the pathogenesis of hepatic fibrosis in NASH.

Chemokine ligand 20 (CCL20) expression increases with NAFLD stage and hepatic stellate cell activation and is regulated by miR-590-5p.

CCL20 (CC chemokine ligand 20) is emerging as an important regulatory molecule in a pathway common to virus infection, alcoholic hepatitis, and non-alcoholic fatty liver disease (NAFLD) leading to the development of hepatic fibrosis. We previously observed upregulation of CCL20 in patients with NAFLD fibrosis and human hepatic stellate cells (LX-2 cells) in response to lipid loading. To date, the mechanisms mediating the relationship between CCL20 and hepatic fibrogenesis remain unknown. In this study, we sought to characterize the molecular mechanisms by which CCL20 may contribute to fibrogenesis in NAFLD. We observed that CCL20 levels increased with worsening severity of liver histology in NAFLD patients (normal < steatosis < inflammation < fibrosis) and during LX-2 cell activation in a time-dependent manner. We found that treatment of LX-2 cells with CCL20 corresponded with increased levels of CCL20 and ACTA2, and decreased levels of PLAU and SERPINE1, effects mitigated by CCL20 knockdown. We identified a putative binding site for miR-590-5p, which we previously reported to be downregulated in NAFLD fibrosis, in the CCL20 3' untranslated region (3'UTR), and found that exogenous miR-590-5p functionally interacted with the CCL20 3'UTR to downregulate its expression. Transfection of LX-2 hepatic stellate cells with miR-590-5p mimic or silencing RNA resulted in decreased or increased CCL20 levels, respectively. Our results indicate an association between CCL20 and hepatic stellate cell activation that includes modulation of key ECM components and functional interactions with a miRNA previously implicated in NAFLD fibrosis. Together, these findings support a novel mechanism by which CCL20 may promote fibrogenesis in NAFLD.

The Role of Long Non-Coding RNAs (lncRNAs) in the Development and Progression of Fibrosis Associated with Nonalcoholic Fatty Liver Disease (NAFLD).

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of conditions ranging from hepatic steatosis to inflammation (nonalcoholic steatohepatitis or NASH) with or without fibrosis, in the absence of significant alcohol consumption. The presence of fibrosis in NASH patients is associated with greater liver-related morbidity and mortality; however, the molecular mechanisms underlying the development of fibrosis and cirrhosis in NAFLD patients remain poorly understood. Long non-coding RNAs (lncRNAs) are emerging as key contributors to biological processes that are underpinning the initiation and progression of NAFLD fibrosis. This review summarizes the experimental findings that have been obtained to date in animal models of liver fibrosis and NAFLD patients with fibrosis. We also discuss the potential applicability of circulating lncRNAs to serve as biomarkers for the diagnosis and prognosis of NAFLD fibrosis. A better understanding of the role played by lncRNAs in NAFLD fibrosis is critical for the identification of novel therapeutic targets for drug development and improved, noninvasive methods for disease diagnosis.