"The Good, the Bad, and the Ugly" - About Diverse Phenotypes of Hepatic Stellate Cells in the Liver.

Hepatic stellate cells (HSCs) and their activated derivatives, often referred to as myofibroblasts (MFs), play a key role in progression of chronic liver injuries leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Until recently, MFs were considered a homogenous cell type majorly due to lack of techniques that allow complex molecular studies at a single-cell resolution. Recent technical advancements in genetic lineage-tracing models as well as the exponential growth of studies with single-cell transcriptome and proteome analyses have uncovered hidden heterogeneities among the HSC and MF populations in healthy states as well as chronic liver injuries at the various stages of tissue deformation. The identification of different phenotypes along the HSC/MF axis, which either maintain essential liver functions ("good" HSCs), emerge during fibrosis ("bad" HSCs), or even promote hepatocellular carcinoma ("ugly" HSCs), may lay the foundation for targeting a particular MF phenotype as potential treatment for chronic liver injuries.

Bioartificial liver with reprogrammed hepatocytes ready for prime time.

In this issue, Wang et al.1 provide evidence of the pre-clinical as well as the clinical utility of in vitro-generated directly reprogrammed human hepatocytes in bioartificial liver. This approach will help offer patients a more curative surgical therapy for liver cancer and improve survival rates.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.

BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. METHODS: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. RESULTS: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. CONCLUSIONS: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.

MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.

microRNA-93-5p promotes hepatocellular carcinoma progression via a microRNA-93-5p/MAP3K2/c-Jun positive feedback circuit.

Cumulative evidence suggests that microRNAs (miRNAs) promote gene expression in cancers. However, the pathophysiologic relevance of miRNA-mediated RNA activation in hepatocellular carcinoma (HCC) remains to be established. Our previous miRNA expression profiling in seven-paired HCC specimens revealed miR-93-5p as an HCC-related miRNA. In this study, miR-93-5p expression was assessed in HCC tissues and cell lines by quantitative real-time PCR and fluorescence in situ hybridization. The correlation of miR-93-5p expression with survival and clinicopathological features of HCC was determined by statistical analysis. The function and potential mechanism of miR-93-5p in HCC were further investigated by a series of gain- or loss-of-function experiments in vitro and in vivo. We identified that miR-93-5p, overexpressed in HCC specimens and cell lines, leads to poor outcomes in HCC cases and promotes proliferation, migration, and invasion in HCC cell lines. Mechanistically, rather than decreasing target mRNA levels as expected, miR-93-5p binds to the 3'-untranslated region (UTR) of mitogen-activated protein kinase kinase kinase 2 (MAP3K2) to directly upregulate its expression and downstream p38 and c-Jun N-terminal kinase (JNK) pathway, thereby leading to cell cycle progression in HCC. Notably, we also demonstrated that c-Jun, a downstream effector of the JNK pathway, enhances miR-93-5p transcription by targeting its promoter region. Besides, downregulation of miR-93-5p significantly retarded tumor growth, while overexpression of miR-93-5p accelerated tumor growth in the HCC xenograft mouse model. Altogether, we revealed a miR-93-5p/MAP3K2/c-Jun positive feedback loop to promote HCC progression in vivo and in vitro, representing an RNA-activating role of miR-93-5p in HCC development.

MicroRNA-221: A Fine Tuner and Potential Biomarker of Chronic Liver Injury.

The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.

Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells.

OBJECTIVE: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases. DESIGN: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter. RESULTS: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver. CONCLUSION: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.

Hepatocyte-specific suppression of microRNA-221-3p mitigates liver fibrosis.

BACKGROUND & AIMS: Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. METHODS: Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. RESULTS: Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C-C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. CONCLUSIONS: Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis. LAY SUMMARY: Liver fibrosis majorly contributes to mortality resulting from various liver diseases. We discovered a small RNA known as miRNA-221-3p, whose downregulation in hepatocytes results in reduced liver fibrosis. Thus, inhibition of miRNA-221-3p may serve as one of the therapeutic approaches for treatment of liver fibrosis.

The Efficacy and Safety of Rivaroxaban and Dalteparin in the Treatment of Cancer Associated Venous Thrombosis.

Venous thromboembolism (VTE) is a complication of malignancy that is associated with significant mortality. The CLOT trial showed superiority of dalteparin in comparison to warfarin in preventing VTE recurrence. Rivaroxaban has been approved for treatment of deep venous thrombosis (DVT) and pulmonary embolism (PE). In the absence of large randomized trials in the oncology population, the efficacy and safety of rivaroxaban for the treatment of VTE in cancer patients needs to be assessed. A single-center retrospective chart review was conducted to assess the efficacy and safety of rivaroxaban compared with dalteparin in cancer-associated thrombosis. Out of 671 patients identified, 286 patients (107 in the rivaroxaban group and 179 in the dalteparin group) were eligible for analysis. The rivaroxaban group had a rate of VTE recurrence at 6 months of 4.9 versus 11.1% with dalteparin (p = 0.252). The incidence of recurrent DVT at 6 months was lower in patients treated with rivaroxaban (0%) compared with dalteparin (8.2%) at 6 months (p = 0.025). Incidence of recurrent PE in the rivaroxaban group (5%) versus dalteparin group (3.1%) at 6 months was not statistically significant (p = 0.675). No significant difference was identified between the rivaroxaban group and dalteparin group in the rate of major bleeding (2.8 vs. 1.1%, respectively). Rivaroxaban was comparable to dalteparin in prevention of VTE recurrence while having no significant differences with major or minor bleeding.

Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model.

AIM: To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-) mice by homologous-recombination-mediated targeted addition of the Fah gene. METHODS: C57BL/6 Fah∆exon5 mice served as an animal model for human tyrosinaemia type 1 in our study. The vector was created by amplifying human Fah cDNA including the TTR promoter from a lentivirus plasmid as described. The Fah expression cassette was flanked by homologous arms (620 bp and 749 bp long) of the Rosa26 gene locus. Mice were injected with 2.1 × 108 VP of this vector (rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR) via the tail vein. Mice in the control group were injected with 2.1 × 108 VP of a similar vector but missing the homologous arms (rAAV8-TTR.Fah). Primary hepatocytes from Fah-/- recipient mice, treated with our vectors, were isolated and 1 × 106 hepatocytes were transplanted into secondary Fah-/- recipient mice by injection into the spleen. Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice. RESULTS: Here, we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the "safe harbour locus" Rosa26 by recombinant AAV8. Both groups of mice showed long-term survival, weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment. In the group of C57BL/6 Fah∆exon5 mice, which have been transplanted with hepatocytes from a mouse injected with rAAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before, 6 out of 6 mice showed long-term survival, weight gain and FAH positive clusters without need for NTBC treatment. In contrast only 1 out 5 mice, who received hepatocytes from rAAV8-TTR.Fah treated mice, survived and showed few and smaller FAH positive clusters. These results demonstrate that homologous recombination-mediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1 (Fah-/- mice) and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/- recipient mice into secondary Fah-/- recipient mice. This long term therapeutic efficacy is clearly superior to our control mice treated with episomal rAAV8 gene therapy approach. CONCLUSION: HR-mediated rAAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/- mice with superior long-term efficacy compared to episomal rAAV8 therapy in proliferating livers.

Let-7c inhibits cholangiocarcinoma growth but promotes tumor cell invasion and growth at extrahepatic sites.

Cholangiocarcinoma (CCA) is a cancer type with high postoperative relapse rates and poor long-term survival largely due to tumor invasion, distant metastasis, and multidrug resistance. Deregulated microRNAs (miRNAs) are implicated in several cancer types including CCA. The specific roles of the miRNA let-7c in cholangiocarcinoma are not known and need to be further elucidated. In our translational study we show that microRNA let-7c expression was significantly downregulated in human cholangiocarcinoma tissues when compared to adjacent tissues of the same patient. Let-7c inhibited the tumorigenic properties of cholangiocarcinoma cells including their self-renewal capacity and sphere formation in vitro and subcutaneous cancer cell growth in vivo. Ectopic let-7c overexpression suppressed migration and invasion capacities of cholangiocarcinoma cell lines in vitro, however, promoted distant invasiveness in vivo. Furthermore, we found that let-7c regulated the aforementioned malignant biological properties, at least in part, through regulation of EZH2 protein expression and through the DVL3/ß-catenin axis. The miRNA let-7c thus plays an important dual role in regulating tumorigenic and metastatic abilities of human cholangiocarcinoma through mechanisms involving EZH2 protein and the DVL3/ß-catenin axis.

Synthesis of Magnetic-Nanoparticle/Ansamitocin Conjugates-Inductive Heating Leads to Decreased Cell Proliferation In Vitro and Attenuation Of Tumour Growth In Vivo.

Conjugates based on nanostructured, superparamagnetic particles, a thermolabile linker and a cytotoxic maytansinoid were developed to serve as a model for tumour-selective drug delivery and release. It combines chemo- with thermal therapy. The linker-modified toxin was prepared by a combination of biotechnology and semisynthesis. Drug release was achieved by hyperthermia through an external oscillating electromagnetic field that induces heat inside the particles. Efficacy of this release concept was demonstrated both for cancer cell proliferation in vitro, and for tumour growth in vivo, in a xenograft mouse model. Biocompatibility studies for these magnetic-nanoparticle/ansamitocin conjugates complement this work.

MYC-driven inhibition of the glutamate-cysteine ligase promotes glutathione depletion in liver cancer.

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC-driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC-driven tumors. We find that fewer glutamine-derived carbons are incorporated into GSH in tumor tissue relative to non-tumor tissue. Expression of GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced microRNA miR-18a. Inhibition of miR-18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC-driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC-dependent attenuation of GCLC by miR-18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.

A Bio-Chemosynthetic Approach to Superparamagnetic Iron Oxide-Ansamitocin Conjugates for Use in Magnetic Drug Targeting.

A combination of mutasynthesis using a mutant strain of A. pretiosum blocked in the biosynthesis of amino-hydroxybenzoic acid (AHBA) and semisynthesis relying on a Stille cross-coupling step provided access to new ansamitocin derivatives of which one was attached by a thermolabile linker to nanostructured iron oxide particles. When exposed to an oscillating electromagnetic field the resulting iron oxide/ansamitocin conjugate 19 heats up in an aqueous suspension and the ansamitocin derivative 16 is released by means of a retro-Diels-Alder reaction. It exerts strong antiproliferative activity (IC50 =4.8 ng mg-1 ) in mouse fibroblasts. These new types of conjugates have the potential for combating cancer through hyperthermia and chemotherapy using an electromagnetic external trigger.

MicroRNA-125b-5p mimic inhibits acute liver failure.

The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF.