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Non-alcoholic fatty liver disease ( NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis, which can progress to fibrosis, cirrhosis and, ultimately, hepatocellular carcinoma. Despite being one of the most common chronic liver diseases, NAFLD pathogenesis remains largely unknown. In this review, we discuss the key molecular mechanisms involved in NAFLD development and progression, focusing on the emerging role of micro RNAs. NAFLD is intrinsically related to obesity and the metabolic syndrome. Changes in lipid metabolism increase free fatty acids in blood, which in turn induces peripheral insulin resistance and increases oxidative and endoplasmic reticulum stress. Although not yet considered in the diagnosis of NAFLD, recent reports also reinforce the crucial role of apoptosis in disease progression via activation of either death receptor or mitochondrial pathways and p53. In addition, the role of gut microbiota and the gut-liver axis has been recently associated with NAFLD. Finally, there is an accumulating and growing body of evidence supporting the role of micro RNAs in NAFLD pathogenesis and progression, as well as hinting at their use as biomarkers or therapeutic tools. The ultimate goal is to review different molecular pathways that may underlie NAFLD pathogenesis in the hope of finding targets for new and efficient therapeutic interventions. [ABSTRACT FROM AUTHOR]

Background and Aims: The mechanisms governing the progression of non‐alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa‐miRNA‐21‐5p in NASH‐related hepatocarcinogenesis. Methods: Hepatic hsa‐miR‐21‐5p expression was evaluated in two cohorts of patients with biopsy‐proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD‐HCC). Serum/liver metabolomic profiles were correlated with hsa‐miR‐21‐5p in NAFLD obese patients. Wild‐type (WT) and Mir21 KO mice were fed a choline‐deficient, amino acid‐defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH‐HCC, respectively. Results: In obese individuals, hsa‐miR‐21‐5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA‐fed WT mice displayed increased hepatic mmu‐miR‐21‐5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre‐neoplastic nodules, hyperplastic foci and deregulated cancer‐related pathways. Mir21 KO mice exhibited peroxisome‐proliferator‐activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro‐inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre‐neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH‐associated carcinogenesis. The hsa‐miRNA‐21‐5p/PPARα pathway was similarly deregulated in patients with HCC‐ or NASH‐related HCC, correlating with HCC markers and worse prognosis. Conclusions: Hsa‐miR‐21‐5p is a key inducer of whole‐spectrum NAFLD progression, from simple steatosis to NASH and NASH‐associated carcinogenesis. The inhibition of hsa‐miR‐21‐5p, leading to a pro‐metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro , in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1 +/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9- NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1 +/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors. [Display omitted] • NAE1-dependent protein NEDDylation is increased in human cholangiocarcinoma. • NEDDylation contributes to cholangiocarcinogenesis favoring pro-survival mechanisms. • Abnormal NEDDylation alters the cholangiocarcinoma secretome, impacting on the stroma crosstalk. • Molecular and pharmacological targeting of NAE1 halts experimental tumor growth. • NAE1 inhibition with pevonedistat is a promising therapy for patients with cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) pathogenesis associates with intramyocellular lipid deposition and mitochondrial dysfunction. microRNAs (miRs), including pro-apoptotic miR-34a, are modulated during disease progression in liver tissue and plasma. We aimed to investigate the functional role of the miR-34a/SIRT1:AMP-activated protein kinase (AMPK) pathway in modulating local mitochondrial dysfunction in the skeletal muscle of human and experimental non-alcoholic steatohepatitis. Muscle biopsies were obtained from morbid obese NAFLD patients undergoing bariatric surgery. C57BL/6N mice were fed different NAFLD-inducing diets and C2C12 muscle cells incubated with palmitic acid (PA) in the presence or absence of an AMPK activator, or upon miR-34a functional modulation. Several muscle miRNAs, including miR-34a, were found increased with human NAFLD progression. Activation of the miR-34a/SIRT1:AMPK pathway, concomitant with impairment in insulin signalling mediators and deregulation of mitochondrial-shaping proteins, was evident in C2C12 cells incubated with PA, as well as in the skeletal muscle of all three diet-induced NAFLD mice models. Functional studies established the association between miR-34a- and PA-induced muscle cell deregulation. Of note, activation of AMPK almost completely prevented miR-34a- and PA-induced cellular stress. In addition, the miR-34a/SIRT1:AMPK pathway and mitochondrial dynamics dysfunction were also found amplified in muscle of human NAFLD. Finally, muscle miR-34a expression and mitofusin 2 (Mfn2) protein levels correlated with hallmarks of NAFLD and disease progression. Our results indicate that activation of the miR-34a/SIRT1:AMPK pathway leads to mitochondrial dynamics dysfunction in skeletal muscle of human and experimental NAFLD, representing an appealing prospective target in metabolic syndrome. Key messages: Skeletal muscle microRNAs are modulated during NAFLD progression. Palmitic acid-induced muscle cell dysfunction occurs, at least in part, through activation of the miR-34a/SIRT1:AMPK pathway. miR-34a/SIRT1:AMPK activation associates with mitochondria dynamics dysfunction in human NAFLD. [ABSTRACT FROM AUTHOR]

Abstract: Background & Aims: Ursodeoxycholic acid (UDCA) is a secondary hydrophilic bile acid (BA) used as therapy for a range of hepatobiliary diseases. Its efficacy in non‐alcoholic fatty liver disease (NAFLD) is still under debate. Here, we aimed to decipher molecular mechanisms of UDCA in regulating endoplasmic reticulum (ER) homeostasis, apoptosis and oxidative stress in morbidly obese patients. Methods: In this randomized controlled pharmacodynamic study, liver and serum samples from 40 well‐matched morbidly obese NAFLD‐patients were analysed. Patients received UDCA (20 mg/kg/d) or no treatment 3 weeks before samples were obtained during bariatric surgery. Results: Patients treated with UDCA displayed higher scoring of steatosis (S), activity (A) and fibrosis (F), the so called SAF‐scoring. UDCA partially disrupted ER homeostasis by inducing the expression of the ER stress markers CHOP and GRP78. However, ERDJ4 and sXBP1 levels were unaffected. Enhanced CHOP expression, a suggested pro‐apoptotic trigger, failed to induce apoptosis via BAK and BAX in the UDCA treated group. Potentially pro‐apoptotic miR‐34a was reduced in the vesicle‐free fraction in serum but not in liver after UDCA treatment. Thiobarbituric acid reactive substances, 4‐hydroxynonenal and mRNA levels of several oxidative stress indicators remained unchanged after UDCA treatment. Conclusion: Our data suggest that UDCA treatment has ambivalent effects in NAFLD patients. While increased SAF‐scores and elevated CHOP levels may be disadvantageous in the UDCA treated cohort, UDCA's cytoprotective properties potentially changed the apoptotic threshold as reflected by absent induction of pro‐apoptotic triggers. UDCA treatment failed to improve the oxidative stress status in NAFLD patients. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of liver lesions, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), that may further progress to cirrhosis. MASLD is estimated to affect more than one third of the general population and it represents a risk factor for end-stage liver failure and liver cancer, substantially contributing to liver-related morbidity and mortality. Although the pathogenesis of MASLD is incompletely understood, it is known to consist of a multifactorial process influenced by extrinsic and intrinsic factors such as metabolic, environmental and demographic features, gut microbiota and genetics. Dysregulation of both extracellular and intracellular lipid composition is known to promote the generation of toxic lipid species, thereby triggering lipotoxicity and cellular stress. These events ultimately lead to the activation of distinct cell death pathways, resulting in inflammation, fibrogenesis and, eventually, carcinogenesis. In this manuscript, we provide a comprehensive review of the role of lipotoxicity during MASLD pathogenesis, discussing the most relevant lipid species and related molecular mechanisms, summarizing the cell type-specific effects and highlighting the most promising putative therapeutic strategies for modulating lipotoxicity and lipid metabolism in MASLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Current therapies for the treatment of alcohol-related liver disease (ALD) have proven largely ineffective. Patients relapse and the disease progresses even after liver transplantation. Altered epigenetic mechanisms are characteristic of alcohol metabolism given excessive acetate and NAD depletion and play an important role in liver injury. In this regard, novel therapeutic approaches based on epigenetic modulators are increasingly proposed. MicroRNAs, epigenetic modulators acting at the post-transcriptional level, appear to be promising new targets for the treatment of ALD., Methods: MiR-873-5p levels were measured in 23 liver tissue from Patients with ALD, and GNMT levels during ALD were confirmed using expression databases (transcriptome n = 62, proteome n = 68). High-resolution proteomics and metabolomics in mice following the Gao-binge model were used to investigate miR-873-5p expression in ALD. Hepatocytes exposed to 50 mM alcohol for 12 h were used to study toxicity. The effect of anti-miR-873-5p in the treatment outcomes of ALD was investigated., Results: The analysis of human and preclinical ALD samples revealed increased expression of miR-873-5p in the liver. Interestingly, there was an inverse correlation with NNMT, suggesting a novel mechanism for NAD depletion and aberrant acetylation during ALD progression. High-resolution proteomics and metabolomics identified miR-873-5p as a key regulator of NAD metabolism and SIRT1 deacetylase activity. Anti-miR-873-5p reduced NNMT activity, fuelled the NAD salvage pathway, restored the acetylome, and modulated the levels of NF-κB and FXR, two known SIRT1 substrates, thereby protecting the liver from apoptotic and inflammatory processes, and improving bile acid homeostasis., Conclusions: These data indicate that targeting miR-873-5p, a repressor of GNMT previously associated with NAFLD and acetaminophen-induced liver failure. is a novel and attractive approach to treating alcohol-induced hepatoxicity., Impact and Implications: The role of miR-873-5p has not been explicitly examined in the progression of ALD, a pathology with no therapeutic options. In this study, inhibiting miR-873-5p exerted hepatoprotective effects against ALD through rescued SIRT1 activity and consequently restored bile acid homeostasis and attenuated the inflammatory response. Targeting hepatic miR-873-5p may represent a novel therapeutic approach for the treatment of ALD., Competing Interests: MLMC advises for Mitotherapeutix LLC. All other authors have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Author(s).)