miR-21-5p promotes NASH-related hepatocarcinogenesis.

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.

Impact of aging on primary liver cancer: epidemiology, pathogenesis and therapeutics.

Aging involves progressive physiological and metabolic reprogramming to adapt to gradual deterioration of organs and functions. This includes mechanisms of defense against pre-malignant transformations. Thus, certain tumors are more prone to appear in elderly patients. This is the case of the two most frequent types of primary liver cancer, i.e., hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Accordingly, aging hallmarks, such as genomic instability, telomere attrition, epigenetic alterations, altered proteostasis, mitochondrial dysfunction, cellular senescence, exhaustion of stem cell niches, impaired intracellular communication, and deregulated nutrient sensing can play an important role in liver carcinogenesis in the elders. In addition, increased liver fragility determines a worse response to risk factors, which more frequently affect the aged population. This, together with the difficulty to carry out an early detection of HCC and iCCA, accounts for the late diagnosis of these tumors, which usually occurs in patients with approximately 60 and 70 years, respectively. Furthermore, there has been a considerable controversy on what treatment should be used in the management of HCC and iCCA in elderly patients. The consensus reached by numerous studies that have investigated the feasibility and safety of different curative and palliative therapeutic approaches in elders with liver tumors is that advanced age itself is not a contraindication for specific treatments, although the frequent presence of comorbidities in these individuals should be taken into consideration for their management.

Isolation of Mitochondria from Liver and Extraction of Total RNA and Protein: Analyses of miRNA and Protein Expression.

Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. DESIGN: RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3-/-) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. RESULTS: RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3-/- mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3-/- mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in Ripk3-/- mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis. CONCLUSION: Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.

Liquid Biopsies in Hepatocellular Carcinoma: Are We Winning?

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and the third most common cause of cancer-related death. One of the major problems faced by researchers and clinicians in this area is the lack of reliable disease biomarkers, which would allow for an earlier diagnosis, follow-up or prediction of treatment response, among others. In this regard, the "HCC circulome", defined as the pool of circulating molecules in the bloodstream derived from the primary tumor, represents an appealing target, the so called liquid biopsy. Such molecules encompass circulating tumor proteins, circulating tumor cells (CTCs), extracellular vesicles (EVs), tumor-educated platelets (TEPs), and circulating tumor nucleic acids, namely circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA). In this article, we summarize recent findings highlighting the promising role of liquid biopsies as novel potential biomarkers in HCC, emphasizing on its clinical performance.

Skeletal muscle miR-34a/SIRT1:AMPK axis is activated in experimental and human non-alcoholic steatohepatitis.

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.

miRNA-21 ablation protects against liver injury and necroptosis in cholestasis.

Inhibition of microRNA-21 (miR-21) prevents necroptosis in the mouse pancreas. Necroptosis contributes to hepatic necro-inflammation in the common bile duct ligation (BDL) murine model. We aimed to evaluate the role of miR-21 in mediating deleterious processes associated with cholestasis. Mechanistic studies established a functional link between miR-21 and necroptosis through cyclin-dependent kinase 2-associated protein 1 (CDK2AP1). miR-21 expression increased in the liver of primary biliary cholangitis (PBC) patients and BDL wild-type (WT) mice at both 3 and 14 days. Notably, under BDL, miR-21 -/- mice displayed decreased liver injury markers in serum compared with WT mice, accompanied by reduced hepatocellular degeneration, oxidative stress and fibrosis. Hallmarks of necroptosis were decreased in the liver of BDL miR-21 -/- mice, via relieved repression of CDK2AP1. Further, miR-21 -/- mice displayed improved adaptive response of bile acid homeostasis. In conclusion, miR-21 ablation ameliorates liver damage and necroptosis in BDL mice. Inhibition of miR-21 should arise as a promising approach to treat cholestasis.

miR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice.

This corrects the article DOI: 10.1038/cddis.2017.172.

miR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice.

microRNAs were recently suggested to contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a disease lacking specific pharmacological treatments. In that regard, nuclear receptors are arising as key molecular targets for the treatment of nonalcoholic steatohepatitis (NASH). Here we show that, in a typical model of NASH-associated liver damage, microRNA-21 (miR-21) ablation results in a progressive decrease in steatosis, inflammation and lipoapoptosis, with impairment of fibrosis. In a complementary fast food (FF) diet NASH model, mimicking features of the metabolic syndrome, miR-21 levels increase in both liver and muscle, concomitantly with decreased expression of peroxisome proliferator-activated receptor α (PPARα), a key miR-21 target. Strikingly, miR-21 knockout mice fed the FF diet supplemented with farnesoid X receptor (FXR) agonist obeticholic acid (OCA) display minimal steatosis, inflammation, oxidative stress and cholesterol accumulation. In addition, lipoprotein metabolism was restored, including decreased fatty acid uptake and polyunsaturation, and liver and muscle insulin sensitivity fully reinstated. Finally, the miR-21/PPARα axis was found amplified in liver and muscle biopsies, and in serum, of NAFLD patients, co-substantiating its role in the development of the metabolic syndrome. By unveiling that miR-21 abrogation, together with FXR activation by OCA, significantly improves whole body metabolic parameters in NASH, our results highlight the therapeutic potential of nuclear receptor multi-targeting therapies for NAFLD.

Activation of necroptosis in human and experimental cholestasis.

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3-/-) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3-/- mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

Circulating microRNAs as Potential Biomarkers in Non-Alcoholic Fatty Liver Disease and Hepatocellular Carcinoma.

Obesity and metabolic syndrome are growing epidemics worldwide and greatly responsible for many liver diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD often progresses to cirrhosis, end-stage liver failure and hepatocellular carcinoma (HCC), the most common primary liver cancer and one of the leading causes for cancer-related deaths globally. Currently available tools for the diagnosis of NAFLD staging and progression towards HCC are largely invasive and of limited accuracy. In light of the need for more specific and sensitive noninvasive molecular markers, several studies have assessed the potential of circulating microRNAs (miRNAs) as biomarkers of liver injury and hepatocarcinogenesis. Indeed, extracellular miRNAs are very stable in the blood, can be easily quantitated and are differentially expressed in response to different pathophysiological conditions. Although standardization procedures and larger, independent studies are still necessary, miRNAs constitute promising, clinically-useful biomarkers for the NAFLD-HCC spectrum.

Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis.

MicroRNAs (miRNAs/miRs) are key regulators of liver metabolism, while toxic bile acids participate in the development of several liver diseases. We previously demonstrated that deoxycholic acid (DCA), a cytotoxic bile acid implicated in the pathogenesis of non-alcoholic fatty liver disease, inhibits miR-21 expression in hepatocytes. Here, we investigated the mechanisms by which DCA modulates miR-21 and whether miR-21 contributes for DCA-induced cytotoxicity. DCA inhibited miR-21 expression in primary rat hepatocytes in a dose-dependent manner, and increased miR-21 pro-apoptotic target programmed cell death 4 (PDCD4) and apoptosis. Both miR-21 overexpression and PDCD4 silencing hampered DCA-induced cell death. Further, DCA decreased NF-κB activity, shown to represent an upstream mechanism leading to modulation of the miR-21/PDCD4 pathway. In fact, NF-κB overexpression or constitutive activation halted miR-21-dependent apoptosis by DCA while opposite results were observed upon NF-κB inhibition. In turn, DCA-induced oxidative stress resulted in caspase-2 activation and NF-κB/miR-21 inhibition, in a PIDD-dependent manner. Finally, modulation of the NF-κB/miR-21/PDCD4 pro-apoptotic pathway by DCA was also shown to occur in the rat liver in vivo. These signalling circuits may constitute appealing targets for bile acid-associated liver pathologies.

Revisiting the metabolic syndrome and paving the way for microRNAs in non-alcoholic fatty liver disease.

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 microRNAs. 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 microRNAs 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.

c-Jun N-terminal kinase 1/c-Jun activation of the p53/microRNA 34a/sirtuin 1 pathway contributes to apoptosis induced by deoxycholic acid in rat liver.

MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose- and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism- and apoptosis-related liver pathologies.