Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis

BACKGROUND: It has been well documented that alcohol and its metabolites induce injury and inflammation in the liver. However, there is no potential biomarker to monitor the extent of liver injury in alcoholic hepatitis patients. MicroRNAs (miRNAs) are a class of non-coding RNAs that are involved in various physiologic and pathologic processes. In the circulation, a great proportion of miRNAs is associated with extracellular vesicles (EVs)/exosomes. Here, we hypothesized that the exosome-associated miRNAs can be used as potential biomarkers in alcoholic hepatitis (AH). METHODS: Exosomes were isolated from sera of alcohol-fed mice or pair-fed mice, and plasma of alcoholic hepatitis patients or healthy controls by ExoQuick. The exosomes were characterized by transmission electron microscopy and Western blot and enumerated with a Nanoparticle Tracking Analysis system. Firefly microRNA Assay was performed on miRNA extracted from mice sera. TaqMan microRNA assay was used to identify differentially expressed miRNAs in plasma of cohort of patients with AH versus controls followed by construction of receiver operating characteristic (ROC) curves to determine the sensitivity and specificity of the candidates. RESULTS: The total number of circulating EVs was significantly increased in mice after alcohol feeding. Those EVs mainly consisted of exosomes, the smaller size vesicle subpopulation of EVs. By performing microarray screening on exosomes, we found nine inflammatory miRNAs which were deregulated in sera of chronic alcohol-fed mice compared to controls including upregulated miRNAs: miRNA-192, miRNA-122, miRNA-30a, miRNA-744, miRNA-1246, miRNA 30b and miRNA-130a. The ROC analyses indicated excellent diagnostic value of miRNA-192, miRNA-122, and miRNA-30a to identify alcohol-induced liver injury. We further validated findings from our animal model in human samples. Consistent with the animal model, total number of EVs, mostly exosomes, was significantly increased in human subjects with AH. Both miRNA-192 and miRNA-30a were significantly increased in the circulation of subjects with AH. miRNA-192 showed promising value for the diagnosis of AH. CONCLUSION: Elevated level of EVs/exosomes and exosome-associated miRNA signature could serve as potential diagnostic markers for AH. In addition to the biomarker diagnostic capabilities, these findings may facilitate development of novel strategies for diagnostics, monitoring, and therapeutics of AH

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis

The Mechanistic Role and Therapeutic Potential of microRNA-122 in Alcoholic Liver Disease: A Dissertation

Chronic alcohol use results in accelerated liver injury, leading to alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, due to the complex nature of this disease process, a central, druggable mechanism has remained elusive. microRNAs are potent post-transcriptional regulators of gene expression. A single miRNA has the ability to regulate hundreds of pathways simultaneously, defining cellular fate and function. microRNA-122 (miR-122), the most abundant miRNA in hepatocytes, has a demonstrated role as an tumor suppressor, regulator of hepatocyte metabolism, and hepatic differentiation. In this dissertation I demonstrate the role of miR-122 on alcoholic liver disease (ALD) pathogenesis over four parts. In chapter II, I will demonstrate chronic alcoholic patients, free of neoplastic changes, have a reduction of miR-122 and that this miRNA regulates HIF-1α, a determinant of ALD pathogenesis. In chapter III, using hepatocytetropic adeno-associated virus 8 (AAV8) vector, I demonstrate that miR-122 inhibition mimics ALD pathogenesis, and furthermore, using hepatocyte-specific HIF-1α-null (HIF1hepKO) mice that this phenomenon is HIF-1α dependent. Given this finding, in chapter IV, I demonstrate that ectopic expression of miR-122 in vivo can reverse alcoholinduced liver damage, steatosis, and inflammation by directly targeting HIF-1α. Finally, in chapter V, I present evidence that alcohol-induced dysregulation of grainyhead-like proteins 1 and 2 (GRHL2), mediate the inhibition of miR-122 at the transcriptional level. These findings dissect a novel mechanistic regulatory axis of miR-122 and indicate a potential opportunity for restoration of miR-122 as a therapy in early ALD.MD/Ph

MicroRNAs in alcoholic liver disease

Alcoholic liver disease (ALD) is characterized by hepatocyte damage, inflammatory cell activation and increased intestinal permeability leading to the clinical manifestations of alcoholic hepatitis. Selected members of the family of microRNAs are affected by alcohol, resulting in an abnormal miRNA profile in the liver and circulation in ALD. Increasing evidence suggests that mRNAs that regulate inflammation, lipid metabolism and promote cancer are affected by excessive alcohol administration in mouse models of ALD. This communication highlights recent findings in miRNA expression and functions as they relate to the pathogenesis of ALD. The cell-specific distribution of miRNAs, as well as the significance of circulating extracellular miRNAs, is discussed as potential biomarkers. Finally, the prospects of miRNA-based therapies are evaluated in ALD

MicroRNAs in alcoholic liver disease

Alcoholic liver disease (ALD) is characterized by hepatocyte damage, inflammatory cell activation and increased intestinal permeability leading to the clinical manifestations of alcoholic hepatitis. Selected members of the family of microRNAs are affected by alcohol, resulting in an abnormal miRNA profile in the liver and circulation in ALD. Increasing evidence suggests that mRNAs that regulate inflammation, lipid metabolism and promote cancer are affected by excessive alcohol administration in mouse models of ALD. This communication highlights recent findings in miRNA expression and functions as they relate to the pathogenesis of ALD. The cell-specific distribution of miRNAs, as well as the significance of circulating extracellular miRNAs, is discussed as potential biomarkers. Finally, the prospects of miRNA-based therapies are evaluated in ALD