Hepatology Basic Science

Abstract

Journal of Gastroenterology and HepatologyVolume 33, Issue S2 p. 27-33 Supplement ArticleFree Access Hepatology Basic Science First published: 06 September 2018 https://doi.org/10.1111/jgh.14392AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Investigation of ischemia–reperfusion injury in an in vitro hepatic steatosis model R Baidya1,2, LA Jaskowski1,2, DHG Crawford1,2 and KR Bridle1,2 1Gallipoli Medical Research Institute, Brisbane, Queensland, Australia; 2School of Clinical Medicine, University of Queensland, Brisbane, Queensland, Australia Introduction: The scarcity of donor livers has triggered efforts to expand the existing donor pool by using marginal donor livers, such as steatotic livers. However, steatotic donor livers are at a high risk of primary graft non-function, early allograft dysfunction, and graft loss due to their enhanced susceptibility to ischemia–reperfusion (I/R) injury. Necroptosis is a novel form of cell death and has been implicated in I/R injury. Receptor-interacting protein kinase 3 (RIPK3) is thought to be instrumental in the execution of necroptosis. However, necroptosis and RIPK3 have not been fully examined in steatotic liver undergoing I/R injury. In this study, we developed an in vitro hepatic steatosis model undergoing I/R injury to further study mechanisms of cell death. Methods: AML-12 cells were cultured in media containing increasing concentrations (0.25, 0.5, 1.0, 2.0) mM of free fatty acid (FFA) for 24 hours to induce hepatic steatosis. Further, FFA-treated cells were subjected to oxygen-glucose deprivation (OGD) conditions by culturing in glucose-free media under hypoxic conditions (1% O2, 5% CO2, and 94% N2) for 12 hours to mimic ischemia. Oil Red O staining was performed to assess the hepatocellular steatosis. Protein expression was detected by western blot analysis, and quantitative polymerase chain reaction was used for mRNA expression quantification. Cell viability was determined by CellTiter-Blue Cell Viability Assay (Promega). Results: A dose-dependent increase in fat accumulation was observed after 24 hours of FFA treatment. There was no significant decrease in cell viability after FFA exposure (P = 0.17). A concentration of 2 mM FFA was considered to be optimal as the cells maintained viability and FFA deposition even after 48 hours of FFA exposure. The hypoxia-sensitive genes, solute carrier family 2, facilitated glucose transporter member 1 (Slca1), and vascular endothelial growth factor (Vegf) were increased (2.6- and 2.7-fold, respectively) after OGD. Treatment with FFA + OGD reduced cell viability after 12 hours of OGD (P < 0.01). RIPK3 protein was significantly upregulated in OGD-treated cells compared with control FFA-treated cells (2.4-fold; P = 0.01). Lack of cleaved-CASPASE3 expression indicated apoptosis was not an active pathway in our model. Nuclear factor NF-κB, which plays a role in regulating the DNA damage-repairing system, decreased in OGD-treated cells (4.6-fold). Similarly, phosphoglycerate mutase family 5 (Pgam5), a gene that protects the cells from necroptosis, was downregulated in OGD-treated cells (1.5-fold). Conclusion: Our findings suggest that necroptosis may contribute to I/R injury in our in vitro model. Future studies will investigate the use of RIPK3 inhibition during the re-oxygenation stage as a therapeutic agent to reduce the consequences of I/R injury. In conclusion, these findings suggest our model may be used to study the cell death pathways active during steatosis and hepatic I/R injury. H-Ferritin and iron activate inflammatory pathways in adipocytes LA Jaskowski1,2, KR Bridle1,2, LJ Britton1,2, A Jayachandran1,2, GA Ramm3 and DHG Crawford1,2 1Gallipoli Medical Research Institute, Brisbane, Queensland, Australia; 2School of Clinical Medicine, University of Queensland, Brisbane, Queensland, Australia; 3QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia Introduction: There is an increasing understanding that adipose tissue dysfunction is an important contributor to hepatic injury in non-alcoholic steatohepatitis and that iron may play a role in exacerbating adipocyte dysfunction. Our previous studies have demonstrated that H-ferritin activated iron-independent inflammatory pathways in hepatic stellate cells, and iron treatment reduced adiponectin secretion in an adipocyte cell line. Thus, the aim of this study was to determine the effect of H-ferritin and iron treatment on inflammatory pathways and adipokine secretion in adipocytes. Methods: NIH 3T3 cells were cultured in differentiation media for 3 days, then cultured in growth media for 5 days before treatment with 0–20 nM H-chain ferritin for 0–240 min or 100 μM ferric ammonium citrate (FAC) for 24 h. Cell viability was determined by MTS assay. Gene expression analysis of chemokine (CC motif) ligand 5 (CCL5 or RANTES), interleukin-6 (IL-6), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), and NOS2 (inducible nitric oxide synthase) was used to determine involvement of inflammatory pathways. Adipokine expression (adiponectin, resistin, and leptin) was examined by quantitative polymerase chain reaction. Results: Treatment of NIH 3T3 cells with H-ferritin or FAC had no impact on viability of the cells at any dose or time-point studied. H-ferritin treatment resulted in time- and dose-dependent increases in CCL5, IL-6, IκBα, and NOS2, with maximal changes at 240 min (3.9-, 4.1-, 1.6-, and 22.9-fold, respectively). Likewise, treatment with FAC increased CCL5, IL-6, IκBα, and NOS2 expression (1.1-, 2.1-, 1.3-, and 3.3-fold, respectively). Adipokine expression (adiponectin, leptin, and resistin) was not significantly different after H-ferritin treatment. Adiponectin and leptin expression were significantly decreased by 100 μM FAC (0.4- and 0.2-fold, respectively); however, resistin expression remained unchanged after FAC treatment. Conclusion: Our findings suggest that inflammatory pathways may be active after H-ferritin and FAC treatment of adipocytes. The inflammatory mediators and adipokines examined are known to affect progression of liver injury and development of fibrosis. Our studies may suggest a link between iron, adipocytes, and the potential progression of hepatic injury in liver conditions associated with elevated ferritin concentrations. Of further interest, this study confirms a relationship between iron metabolism and regulation of appetite. Dietary advanced glycation end products play a major role in exacerbating progression of non-alcoholic fatty liver disease to liver fibrosis in diabetic mice HKDH Fernando1, DIG Rajapaksha1, JM Forbes3, PW Angus1,2 and CB Herath1 1Department of Medicine, University of Melbourne, Brisbane, Queensland, Australia; 2Department of Gastroenterology and Hepatology, Austin Health, Melbourne, Victoria, Australia; 3Mater Research, University of Queensland, Brisbane, Queensland, Australia Introduction: Non-alcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population and is now a major cause of liver disease-related premature illness and death in Australia. Advanced glycation end products (AGEs), formed as a result of non-enzymatic reaction between reducing sugars and proteins, lipids, or nucleic acids and acting via RAGE (receptor for advanced glycation end products), have been implicated as a second hit that drives NAFLD to steatohepatitis and liver fibrosis. Western diets often contain high levels of AGEs due to preparation of food at high temperatures. Although endogenous AGE production is increased with diabetes, the impact of high dietary AGE levels on NAFLD progression in diabetes is unknown. Therefore, in this study, we investigated the role of dietary AGEs in NAFLD progression in diabetic mice fed a high-fat, high-cholesterol (HFHC) diet containing high levels of AGEs. Methods: Six-week-old male C57Bl/6 mice were fed an HFHC diet for 40 weeks. A second group of mice was fed the HFHC diet after baking it at 160 °C for 1 hour (HFHC + B) to increase dietary AGE content. Another two groups of mice that were fed on either HFHC or HFHC + B diet were made diabetic at Week 15 by streptozotocin injections (65 mg/kg/bw). Content of N(6)-(carboxymethyl)lysine (CML), the best characterized AGE type, in HFHC and HFHC + B diets was measured by gas chromatography–mass spectrometry. Non-fasting glucose levels were measured at regular intervals in diabetic animals. At 40 weeks, animals were sacrificed, and blood and liver tissue were harvested. Liver function tests were done to investigate liver injury. Cell culture experiments were performed using an immortalized Kupffer cell line (KUP5 cells). Gene expression of RAGE, profibrotic cytokines transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), and proinflammatory cytokines interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), and hepatic stellate cell (HSC) activation marker α-smooth muscle actin (αSMA) was determined by quantitative polymerase chain reaction. Liver histology and fibrosis were determined by hematoxylin and eosin and picrosirius red staining, respectively. Results: CML levels were threefold higher in mice on the HFHC + B diet than those on the HFHC diet. The HFHC diet produced liver steatosis at 40 weeks. Blood glucose levels were significantly increased in diabetic animals. Liver RAGE expression was significantly (P < 0.05) increased by high dietary AGEs (HFHC + B) and diabetes (HFHC + D) compared with that of HFHC-fed non-diabetic mice and further upregulated (P < 0.01) by high dietary AGE intake in diabetic mice (HFHC + B + D). Moreover, both dietary AGEs and diabetes significantly (P < 0.05) increased liver expression of IL-1β, IL-6, TNF-α, MCP-1, and αSMA compared with HFHC-fed mice. Similarly, high dietary AGE intake and diabetes increased (P < 0.05) gene expression of liver endotoxin responsive proteins, CD14 and toll-like receptor-4 (TLR4), and profibrotic cytokines such as CTGF and TGF-β1. Unlike the effect on RAGE expression, however, diabetes did not have an additive effect on the expression of genes described above after feeding with the high AGE diet. While there was moderate fibrosis in HFHC-fed mice compared with that of normal chow-fed mice, there was increased (P < 0.01) liver fibrosis in diabetic (HFHC + D) and non-diabetic high dietary AGE-fed (HFHC + B) mice compared with littermates fed on the HFHC diet (HFHC). However, the profibrotic effect of high dietary AGEs in non-diabetic mice was higher (P < 0.05) than that in diabetic littermates fed on the HFHC diet (HFHC + D). The combination of diabetes with dietary AGEs (HFHC + B + D) did not enhance their profibrotic effect. Treatment of KUP5 cells with AGEs (200 μg/mL) caused an increased (P < 0.05) expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α compared with cells treated with the vehicle. Conclusion: Both dietary AGEs and diabetes have a strong effect in driving NAFLD progression to liver fibrosis. While it is not known whether the effect of diabetes on liver fibrosis is attributable to increased endogenous AGE formation, we conclude that, in this model, dietary AGEs perpetuate liver fibrosis to a greater degree than diabetes alone. This effect may be mediated by direct effects on liver resident macrophages (Kupffer cells). Effects of miRNA-25-3p overexpression on cross-talk between transforming growth factor-β, Notch, and Wnt signaling in hepatic stellate cells B Genz1,2,3, MA Coleman1, KM Irvine2,3, JR Kutasovic3,4, M Miranda4, F Al-Ejeh4, DA Calvopina1, A Weis1, N Cloonan5, H Robinson6, MM Hill6 and GA Ramm1,3 1Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 2Mater Research, Translational Research Institute, Brisbane, Queensland, Australia; 3Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia; 4Personalised Medicine Team, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 5Genomic Biology Lab, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 6Precision and Systems Biomedicine, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia Introduction: Hepatic stellate cells (HSCs) are considered to be the main source of extracellular matrix in liver fibrosis. During chronic liver injury, HSCs become activated and transdifferentiate into pro-fibrotic myofibroblasts. This process is associated with major changes in gene expression, as well as intracellular signaling, and can potentially be regulated by microRNAs (miRNA). In a recent study, we found miRNA-25-3p (miR-25) to be upregulated in serum of children with cystic fibrosis (CF) who have no evidence of liver disease, compared with children with CF liver disease and healthy individuals. In this study, we investigated the role of miR-25 in HSC biology and on the development of liver disease. Methods: MiR-25 expression was measured in the human HSC cell line LX-2 and primary murine HSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization. The effect of miR-25 overexpression on HSCs was examined using qRT-PCR, NanoString nCounter mRNA analysis, and western blot 24, 48 and 72 hours after transfection of LX-2 cells with miR-25 or control miRNA. Smad phosphorylation and collagen1α1 induction in response to transforming growth factor (TGF)-β in miR-25-overexpressing LX-2 cells were measured by qRT-PCR and western blot. Putative miR-25 mRNA targets in LX-2 cells were identified by pulldown experiments using biotinylated miR-25 duplexes and validated by luciferase reporter assay and qRT-PCR. Results: MiR-25 was expressed in LX-2 cells, as well as in activated primary HSCs. MiR-25 overexpression inhibited mRNA expression of TGF-β and its type 1 receptor (TGFBR1) in LX-2 cells. MiR-25 overexpression inhibited TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction. Pulldown experiments revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as targets of miR-25 in HSCs, and expression of Notch signaling pathway components was downregulated in miR-25 overexpressing LX-2 cells. Furthermore, expression of Wnt signaling components (especially WNT5A) was also reduced by miR-25 overexpression. Conclusion: Our data suggest that miR-25 functions as a negative feedback control during HSC activation, reducing the reactivity to TGF-β by regulation of TGFBR1 and the cross-talk between Notch, Wnt, and TGF-β signaling. Therefore, we propose that miR-25 may act as a potential antifibrotic agent in regulating the development of liver disease. Mas-related G protein-coupled receptor type D is a novel therapeutic target to reduce splanchnic vasodilatation in portal hypertension LS Gunarathne1, A Zulli3, T Qaradakhi3, R Smith3, AB Tacey3, PW Angus1,2 and CB Herath1 1Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia; 2Department of Gastroenterology and Hepatology, Austin Health, Melbourne, Victoria, Australia; 3College of Health and Biomedicine, Vectoria University, Melbourne, Victoria, Australia Background and Aims: Splanchnic vasodilatation, which leads to an elevated portal venous inflow in patients with cirrhosis, plays an important role in the pathogenesis of portal hypertension. Our previous studies have suggested that activation of the Mas receptor (MasR) of the so-called alternate arm of the renin angiotensin system (RAS) contributes to this vasodilatation. In the current study, we investigated the effect of blockade of the Mas-related G protein-coupled receptor type D (MrgD), another recently characterized receptor of the RAS, on splanchnic vasodilatation and portal pressure (PP), and compared this with the effects of MasR blockade in cirrhotic animals. Methods: Liver disease was induced in male Sprague Dawley rats by bile duct ligation (BDL) surgery or twice-weekly carbon tetrachloride (CCl4) injections. Two weeks after BDL and 8 weeks after CCl4 injections, the animals received either the MrgD antagonist D-Pro-7-Ang-(1-7) (D-Pro) or MasR antagonist A779 (28 μg/kg bw/h) for 2 weeks via subcutaneously implanted osmotic minipumps. Sham-operated and olive oil-injected healthy rats, and BDL and CCl4 animals receiving saline infusion, served as controls. Two weeks after treatment, catheters were placed in the portal vein and a femoral artery to measure PP and mean arterial pressure (MAP), respectively. The colored microsphere technique was used to calculate splanchnic vascular resistance (SVR), hepatic vascular resistance (HVR), and mesenteric blood flow (MBF). Mesenteric resistance vessels were obtained from a separate group of CCl4-injected cirrhotic rats to study and compare the vascular effects of MrgD blockade with those of MasR blockade. Results: D-Pro and A779 significantly (P < 0.05) reduced PP in both models compared with saline-infused disease controls; however, this reduction was larger (P < 0.05) in CCl4-injected rats treated with MrgD than MasR blockers. This reduction in PP resulted from significantly increased SVR in CCl4 (P < 0.0005) and BDL (P < 0.01) models with D-Pro treatment, and in CCl4 (P < 0.01) and BDL (P < 0.05) models with A799 treatment compared with saline-infused disease controls, although the treatments also increased HVR. In the CCl4 model, the increased SVR with MrgD blockade was much greater (P < 0.05) than that of MasR blockade, leading to a marked reduction in MBF, which was higher (by 15%) in D-Pro- than A779-treated animals. MasR blockade, but not MrgD blockade, produced an off-target effect by elevating MAP (P < 0.05) in BDL rats. In support of the above observations with MrgD blockade, first-order as well as second/third-order mesenteric resistant vessels treated with the MrgD blocker D-Pro showed a profound reduction in vasorelaxation in response to acetylcholine (maximum relaxation of 45% and 13%, respectively), whereas MasR blocker A779 failed to block acetylcholine-induced relaxation. Conclusion: The findings showed profound effects of MrgD blockade on splanchnic vascular resistance, portal blood flow, and PP. Moreover, MrgD but not MasR blockade has splanchnic vasculature-specific effects in cirrhotic rats. Thus, we have identified MrgD as a potential therapeutic target for the design of drugs that can specifically block splanchnic vasodilatation in cirrhotic portal hypertension. Identification of mutations in circulating cell-free tumor DNA as a prognostic biomarker in hepatocellular carcinoma J Howell1, 1,2,3, SR Atkinson1, 1, DJ Pinato1, S Knapp1,4, C Ward1, R Minisini5, ME Burlone5, M Leutner5, M Pirisi5, R Büttner6,7, SA Khan1, M Thursz1, M Odenthal1, 6,7 and R Sharma1, 1 1Department of Surgery and Cancer, Imperial College London, London, UK; 2Centre for Population Health, Burnet Institute, Melbourne, Victoria, Australia; 3Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia; 4Department of Women's Cancer, Institute for Women's Health, University College London, London, UK; 5Department of Translational Medicine, Università degli Studi del Piemonte Orientale “A. Avogadro”, Novara, Italy; 6Institute for Pathology, University Hospital of Cologne and Center of Integrative Oncology, Cologne, Germany; 7Center of Molecular Medicine Cologne, Cologne, Germany 1Equally contributed. Introduction: Hepatocellular carcinoma (HCC) is increasing globally. Prognostic biomarkers are urgently needed to guide treatment and reduce mortality. Circulating cell-free DNA of tumor origin (ctDNA) is a novel, minimally invasive means of determining genetic alterations in cancer. We evaluate the accuracy of ctDNA as a biomarker in HCC. Methods: Plasma cell-free DNA, matched germline DNA, and, where available, HCC tissue DNA were isolated from patients with HCC across BCLC stages (cases, n = 51) and liver cirrhosis (controls, n = 10). Targeted, multiplex polymerase chain reaction ultra-deep sequencing was performed using a liver cancer-specific primer panel for genes ARID1A, ARID2, AXIN1, ATM, CTNNB1, HNF1A, and TP53. Concordance of mutations in plasma ctDNA and HCC tissue DNA from the same patients was determined, and associations with clinical outcomes were analyzed. Results: Plasma cell-free DNA was detected in all samples. Cases with BCLC stage A HCC had lower plasma cell-free DNA levels than those with BCLC stage B, C, or D HCC (median cell-free DNA concentration, 122.89 ng/mL [IQR, 57.17–154.26 ng/mL] compared with 168.21 ng/mL [IQR, 135.46–202.89 ng/mL], P = 0.041). Thirty-one mutations in the seven genes (23 unique mutations) were detected in 18 of 51 HCC patients (35%), with a median of 1.5 mutations per patient (IQR, 1–2). Among eight patients with matched HCC tissue DNA available, all mutations detected in plasma ctDNA were confirmed in HCC DNA. Mutations were most frequently detected in ARID1A (7/51 patients, 13.7%), followed by CTNNB1 (4, 7.8%) and TP53 (4, 7.8%). Conclusion: ctDNA is quantifiable across all HCC stages and allows detection of mutations in key driver genes of hepatic carcinogenesis. This study demonstrates high concordance between mutations detected in plasma ctDNA and those detected in matched HCC tissue. Macrophage colony-stimulating factor promotes regression of advanced fibrosis and liver regeneration in mice B Genz1,2, AD Clouston3, DA Hume2 and KM Irvine2 1QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 2Mater Research Institute, University of Queensland, Brisbane, Queensland, Australia; 3Faculty of Medical and Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia Introduction: Optimal therapeutic approaches to ameliorate liver fibrosis, especially advanced liver disease, may require both reversal of fibrosis and liver regeneration. We hypothesize that appropriate stimulation of macrophage function may achieve both these goals because, despite their clear contribution to fibrogenesis, macrophages also play critical roles in liver regeneration and fibrosis resolution and exhibit a high degree of plasticity in response to their microenvironment. Macrophages differentiated in the presence of macrophage colony-stimulating factor (CSF1) are implicated in wound healing responses, and we have previously shown that a recombinant CSF1-Fc conjugate, which has an extended half-life in vivo compared with the rapid clearance of the native cytokine, promotes liver growth in healthy mice, rats, and pigs. We thus hypothesized that CSF1-Fc therapy would drive matrix remodeling, fibrosis regression, and liver regeneration. We tested this hypothesis using a toxin-induced model of progressive liver injury and fibrosis. Methods: Groups of six wild type, female C57/Bl6 mice were administered thioacetamide (TAA) in drinking water for 8 weeks, followed by cessation of TAA treatment and biweekly administration of 1 mg/kg CSF1-Fc or saline control for 4 weeks. Inflammation, myofibroblast activation, hepatocyte proliferation, and fibrosis were assessed by immuno-/histochemical approaches and whole-liver quantitative polymerase chain reaction (qPCR). Dynamic changes in liver monocyte and macrophage populations were quantified using flow cytometry. The expression of pro-inflammatory and pro-fibrotic mediators and matrix remodeling enzymes was assessed by qPCR. Ethical approval for the study was obtained from the University of Queensland Animal Ethics Committee. Results: Administration of TAA for 8 weeks induced chronic inflammation, myofibroblast activation, and advanced (bridging) fibrosis. Despite rapid resolution of inflammation, myofibroblast activation (disappearance of SMA-positive cells), and Collagen 1A1 (Col1a1) mRNA expression, there was no regression of histological fibrosis (Sirius red staining) 4 weeks after cessation of TAA treatment in control mice. By contrast, biweekly CSF1-Fc treatment significantly reduced hepatic collagen content, which was associated with upregulation of genes encoding matrix remodeling factors such as matrix metalloproteinase (Mmp) 9, Mmp13, and urokinase plasminogen activator (Plau). In addition to fibrolysis, CSF1-Fc treatment increased liver weight more than twofold, which was associated with increased hepatocyte proliferation (Ki67 staining). CSF1-Fc treatment elicited hepatic monocyte infiltration and accumulation of monocyte-derived macrophages and non-classical Ly6CLow monocytes that have previously been shown to promote resolution of liver fibrosis by production of matrix remodeling and growth factors. Conclusions: Our data suggest that CSF1-Fc functions as a “restorative” macrophage switch and may represent a novel therapeutic approach to achieve both remodeling of advanced fibrosis and liver regeneration that is necessary for the restitution of normal liver architecture and function. MicroRNA therapeutics for hepatocellular cancer T Kabir, R Brown, K Richardson, G Yeoh and PJ Leedman Laboratory for Cancer Medicine, Harry Perkins Institute of Medical Research, Centre for Medical Research and the Medical School, University of Western Australia, Perth, Western Australia, Australia Hepatocellular cancer (HCC) is increasing in incidence and has a very poor prognosis. New treatments using new modalities are urgently needed. There have recently been major advances in RNA-based therapeutics (siRNA, miRNA, and antisense oligonucleotides) so that they now offer great potential to treat a range of human disorders, including cancer. RNA-based drugs developed with second-generation chemistry are proving very successful in liver disorders, best illustrated by siRNA targeting of PCSK9, which profoundly reduces low-density lipoprotein cholesterol in patients already taking statins.1 Key to this advancement has been: (i) the enhanced stability afforded by structural modifications to the RNA oligonucleotides; (ii) hepatocyte-specific delivery via the high affinity asialoglycoprotein receptor-GalNAc (N-acetyl galactosamine) interaction; and (iii) dispensing with needing a lipid carrier or vehicle, the latter being a potential cause of therapy-limiting adverse effects. The EGF-receptor (EGFR) and its signaling pathways are aberrantly expressed in HCC and represent an important therapeutic target. We have been developing a microRNA for cancer therapy, miR-7, which is a potent inhibitor of the EGFR signaling pathway in poor prognostic tumors (head and neck cancer, glioma).2 In HCC, miR-7 expression is reduced (typical of a tumor suppressor miRNA), and when miR-7 levels in HCC cells are restored, it results in powerful inhibition of EGFR signaling associated with significant reduction of cell viability in vitro and tumor growth in vivo. In addition to the EGFR pathway, miR-7 targets multiple other receptors and signaling molecules, including TYRO3 (a member of the TAM family of tyrosine kinase receptors) and P-Akt. We have recently generated HCC cell lines that are resistant to sorafenib, the routinely used multi-tyrosine kinase inhibitor for HCC, in an effort to recapitulate the development of acquired resistance in patients with HCC. Interestingly, we found that miR-7 can overcome resistance to sorafenib,3 raising the possibility that miR-7, either alone or in combination with sorafenib, could be a useful approach to combatting HCC drug resistance. Based on these data suggesting miR-7 could be a useful therapy in HCC, we have embarked on a collaborative program with a United States-based RNA therapeutics company to develop novel miR-7 mimics. These new molecules feature second-generation chemistry and are designed to target the liver specifically, removing the need for a lipid vehicle. To date, we have generated novel miR-7 mimics that are at least as potent as the commercially available miR-7, and we are exploring additional modifications to optimize therapeutic efficacy and enhance bioavailability in vivo, while minimizing potential adverse effects. Taken together, these data suggest that miR-7 is a master regulator of several key tyrosine kinase receptors and their downstream signaling pathways in HCC that results in potent inhibition of HCC cell growth and viability. Furthermore, miR-7 can overcome sorafenib resistance, providing additional incentive to develop a novel miR-7 mimic that can be evaluated in early-phase HCC clinical trials. References 1Ray KK, Landmesser U, Leiter LA et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 2017; 376: 1430. 2Kalinowski FC, Giles KM, Candy PA et al. Regulation of epidermal growth factor receptor signaling and erlotinib sensitivity in head and neck cancer cells by miR-7. PLOS One 2012; 7(10): e47067. 3Kabir TD, Ganda C, Brown RM et al. A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma. Hepatology 2018; 67: 216. Immune checkpoint inhibitors and tumor mutation burden as predictive biomarkers in hepatocellular carcinoma R Shrestha1,2, P Prithviraj3, KR Bridle1,2, DHG Crawford1,2, M Anaka4 and A Jayachandran1,2 1Liver Cancer Unit, Gallipoli Medical Research Institute, Greenslopes Private Hospital, Brisbane, Queensland, Australia; 2Faculty of Medicine, Un