Prof Jeanne-Marie Perotin

Gabrielle Wheway

Kamran Tariq

Adnan Azim

Robert A Ridley

Jonathan A Ward

James Pr Schofield

Clair Barber

Peter Howarth

Donna E Davies

Ratko Djukanovic

Vulnerability to acid reflux of the airway epithelium in severe asthma

Keywords: Asthma, Gastroesophageal reflux disease, Airway epithelium, Air liquid interface air-liquid interface CDC: chenodeoxycholic acid DEG: differentially expressed genes FDR: False discovery rate GORD: gastro-oesophageal reflux disease MCP-PAB: multiple challenge protocol with pepsin, acid pH and bile acids PBEC: primary bronchial epithelial cells PPI: proton pump inhibitor TEER: transepithelial electrical resistance TEM: transmission electron microscopy UPR: Unfolded protein response

INTRODUCTION

Severe asthma is associated with multiple co-morbidities, including gastro-oesophageal reflux disease (GORD) which is particularly common and is associated with exacerbation frequency and poor quality of life (1). Until recently, this association was explained by three mechanisms: vagal reflex (2), neuroinflammation (3), and microaspiration directly triggering airway inflammation (4)(5)(6)(7). While studies of reflux in animal models (4)(5)(6)8) and cultures of bronchial epithelial cells (9,10) have shown varying impact of gastro-oesophageal refluxate on mediators of inflammation and airway remodelling, direct in vivo evidence for these mechanisms in patients with asthma has been limited. We recently undertook an in-depth analysis of sputum proteomics in severe asthmatics and identified 11 proteins differentially abundant in patients with GORD, including elevated levels of anti-microbial proteins and reduced levels of proteins involved in systemic inflammatory responses and epithelial integrity (11), providing the first direct evidence that reflux is associated with changes in the microenvironment on the epithelial surface of the airways. Recognising that defective epithelial barrier function, dysregulated repair mechanisms, and modified epithelial immune responses to pathogens and allergens are important features in asthma [START_REF] Davies | Epithelial barrier function and immunity in asthma[END_REF], we further hypothesised that the presence of GORD in severe asthma would significantly influence global epithelial gene expression. Applying unbiased topological data analysis (TDA) of microarray data derived from bronchial brushings, we identified a subset of severe asthmatics with a clinical phenotype defined by obesity, presence of GORD and treatment with proton pump inhibitors (PPI) [START_REF] Perotin | Epithelial dysregulation in obese severe asthmatics with gastro-oesophageal reflux[END_REF], characterized by upregulated airway remodelling signalling and downregulated mechanisms of immune cell recruitment, possibly linked to both bile acid exposure and PPI treatment [START_REF] Perotin | Epithelial dysregulation in obese severe asthmatics with gastro-oesophageal reflux[END_REF].

In the current study, we have sought to elucidate further the underlying mechanisms of GORDassociated dysregulation of the airway epithelium in severe asthma using a combination of in vitro and ex vivo approaches. We developed an in vitro model of GORD in which fully differentiated air-liquid interface (ALI) cultures of primary bronchial epithelial cells were exposed to a multiple challenge protocol using pepsin, acid pH and bile acids (MCP-PAB).

Consistent with our previous in vivo observations [START_REF] Perotin | Epithelial dysregulation in obese severe asthmatics with gastro-oesophageal reflux[END_REF], we observed that ex vivo exposure of epithelial cells to refluxate results in significant structural and functional changes. We then extended our studies using bronchial biopsies and bronchial brushings from severe asthmatics with GORD and confirmed the effects on IL-33 and changes in expression of a selection of genes identified from the in vitro study.

METHODS

Study participants and sample collection

Severe asthmatics (Step 4/5 of BTS/SIGN Guidelines) and healthy control participants were recruited prospectively and assessed for GORD by 24-hr pH/impedance studies. The severe asthmatics were further stratified into those with documented GORD but no PPI treatment, those with documented GORD and PPI treatment and those without GORD. Epithelial cells were harvested by bronchoscopic brushings and processed into RNAlater for subsequent RNAseq analysis or used in primary bronchial epithelial cell (PBEC) culture [START_REF] Xiao | Defective epithelial barrier function in asthma[END_REF]. Bronchial biopsies were also taken and fixed in paraffin for immunohistochemical analyses.

The study was approved by the South-Central Hampshire A, Research Ethics Committee, UK (reference numbers: 13/SC/0182 and 14/WM/1226) and all participants gave their informed consent.

Analysis of the ex vivo effect of a multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB)

Initial dose and time-course studies were conducted with 16HBE cells exposed to MCP-PAB (pepsin and chenodeoxycholic acid at acidic pHs); optimised conditions were subsequently confirmed to be appropriate for fully differentiated air liquid interface (ALI) cultures (see online supplement and Figure E1 for full details). MCP-PAB conditions were applied to the apical epithelial surface for 30 minutes before washing twice. After 4h recovery, epithelial permeability was measured using transepithelial electrical resistance (TEER) and FITC dextran 4kDa [START_REF] Xiao | Defective epithelial barrier function in asthma[END_REF]. Apical supernatants were collected for cytokine measurements. Cells were then lysed with Trizol lysis reagent (Life Technologies, Paisley, UK) and frozen at -80°C until analysis or fixed for immunofluorescence staining or electron microscopy analysis.

Immunostaining and electron microscopy

ALI cultures were fixed with 4% paraformaldehyde and processed for immunofluorescence staining, as previously described [START_REF] Xiao | Defective epithelial barrier function in asthma[END_REF]. The cultures were also processed for transmission electron microscopy (TEM) and analysed for epithelial integrity.

Bronchial biopsies were processed as previously described [START_REF] Wilson | Severe asthma exists despite suppressed tissue inflammation: findings of the U-BIOPRED study[END_REF], embedded into paraffin; sections were stained using goat polyclonal anti-human IL-33 (R&D Systems, Abingdon, UK).

Results were expressed as positive nuclei per total number of epithelial cells.

Cytokine analyses

Interleukin (IL)-8 concentrations in conditioned media were measured using an IL-8 DuoSet ELISA (R&D, Abingdon, UK) while IL-6, TNFα and IL-1α were measured using VPlex (MSD, Maryland, USA).

Analysis of gene expression in epithelial brushings and differentiated cells

Total RNA was extracted from epithelial brushings or cultured cell lysates using miRNeasy Mini Kit and RNase-Free DNase Set (Quiagen, Manchester, UK). cDNA libraries were prepared using NEBNext Ultra (non-stranded) mRNA library prep kit with polyA pulldown for mRNA enrichment. Paired-end 150bp sequencing to a depth of 20M reads (epithelial brushings) or 50M reads (differentiated cells) was performed on an Illumina HiSeq2500 by Novogene Inc (Cambridge, UK). FATSQ files were aligned to human genome build GRCh38 using STARv2.6.0, reads were counted with HTSeq and differential gene expression analysis conducted with edgeR. Details are in the online supplement. Data are deposited in GEO.

Statistical analyses

Paired t-tests were applied to transcriptomic data, while clinical and experimental data were analysed by Kruskal-Wallis, Mann-Whitney U or Student t tests depending on data distribution; p<0.05 was considered significant. False discovery rate (FDR) correction was applied to the transcriptomic data.

RESULTS

Multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) causes epithelial damage and alters barrier and secretory function

To analyse the impact of GORD on the airway epithelium of severe asthmatics, ALI cultures derived from bronchial brushings of participants with severe asthma, GORD and PPI treatment (n=8) and healthy controls (n=5) (Table 1) were exposed for 30 min to MCP-PAB conditions consisting of 50µg/ml of pepsin and 250µM chenodeoxycholic acid (CDC) at pH 5.

MCP-PAB-induced epithelial damage was characterized by TEM as enlargement of intercellular spaces and beginning of cell detachment (Figure E2). MCP-PAB-exposed ALI cultures also had markedly increased ionic and macromolecular permeability, as shown by decreased TEER (Figure 1a) and increased FITC dextran 4kDa passage respectively (Figure 1b), with a significantly greater impact of MCP-PAB on permeability of cultures from severe asthmatic donors compared with healthy controls. Analysis of epithelial tight junctions and adherens junctions in ALI cultures from severe asthmatic donors showed a marked disruption of the junctions in MCP-PAB-exposed cultures (Figure E3).

In addition to having a marked impact on epithelial structure, MCP-PAB caused an increase in the secretion of CXCL8, IL1α, and TNFα (Figure 2). These results were supported by analysis of epithelial gene expression (Figure E4).

Artificial refluxate upregulates unfolded protein responses, damage-responses and epithelial remodelling mechanisms.

To further analyse the mechanisms involved in MCP-PAB-induced epithelial dysregulation in ALI cultures, we analysed mRNA transcriptomes obtained by RNAseq.

Comparison of gene expression in unstimulated ALI cultures showed 147 genes upregulated and 266 downregulated in cultures from severe asthmatics when compared with healthy control (Figure 3, Table E1). Application of MCP-PAB resulted in a profound effect on gene expression, especially in ALI cultures from severe asthmatic donors which had a significantly higher number of differentially expressed genes (DEGs) (n=599) compared to cultures from healthy donors (n=87 DEGs). Amongst the most prominent modulated genes were IL1RL1 (Interleukin 1 Receptor Like 1, the receptor for IL33), CHAC1 (cation transport regulator-like protein 1), involved in oxidative balance and unfolded protein response (UPR), and SERPINB9, a serine protease inhibitor.

Gene ontology analysis (AmiGO) of all MCP-PAB-induced DEGs identified a number of differentially controlled biological processes (p<0.05). Taking a cut-off of 2-fold increase in gene expression, we found 57 processes upregulated in cultures from severe asthmatic donors and 25 in those from healthy donors. In order to identify the processes with the greatest impact, we undertook a further selection of gene expression with a cut-off of 5-fold; this showed 16 processes upregulated in cultures from severe asthmatic donors and 11 in cultures from healthy controls (Table 2).

The most significant enrichment due to MCP-PAB exposure was in the PERK-mediated UPR; this was significant in cultures from both asthmatics and healthy donors but was three times greater in healthy participants. Cultures from healthy individuals exposed to MCP-PAB were also enriched in other stress response processes (Table 2). In contrast, MCP-PAB-exposed epithelial cultures derived from severe asthmatic donors were enriched in EGFR signalling, cell migration and vasculature development, suggesting upregulation of tissue repair and remodelling responses.

Having established MCP-PAB-induced epithelial damage, we next explored the impact of MCP-PAB on damage-signalling. We analysed the damage-associated cytokine IL-33 and found that AR caused increased nuclear IL-33 staining cultures from severe asthmatic donors (Figure 4). We confirmed IL-33 expression in ALI cultures using qPCR (HC, n=5; SA, n=6), and showed that MCP-PAB was associated with a 67% increase in IL-33 expression in SA and a -11% change in HC (p=0.01 for between group comparison).

Comparison of in vitro findings with in vivo epithelial changes in severe asthma with GORD

To determine the relevance of our in vitro findings with epithelial changes in severe asthma with GORD, we first performed IHC for IL-33 using bronchial biopsies from severe asthmatic (n=9) or healthy control subjects without GORD (n=4); the severe asthmatics were sub-grouped as follows: (i) SA with no GORD (n=5), and (ii) SA with documented GORD but who had abstained from their regular PPI treatment for 2 weeks to avoid potential bias of systemic or local (through micro-inhalation) impact of PPI on epithelial gene expression (n=4). As shown in Figure 5, there was a significantly higher number of IL-33 positive nuclei in SA-no GORD compared to healthy controls with a further significant increase in SA-GORD.

Finally, we analysed mRNA transcriptomes of bronchial brushings from SA-GORD no PPI (n=6), SA-no GORD (n=4) and healthy control subjects (n=12). RNAseq analysis identified that of the top 37 genes whose expression was modified in ALI cultures in response to MCP-PAB, 15 were similarly modified ex vivo in patients with GORD (Table 3). Of note, the expression of CHAC1, the top upregulated gene involved in the UPR process which was identified as the main mechanism induced by MCP-PAB in ALI cultures (Table E1), was also increased in bronchial brushings obtained from SA-GORD when compared to SA-no GORD, confirming a similar impact of refluxate on epithelial responses to endoplasmic reticulum stress in vivo.

DISCUSSION

Using a combination of in vitro and ex vivo approaches we have obtained compelling evidence in support of reflux having a significant impact on bronchial epithelial structure and function, with a profound effect on the epithelium of severe asthma patients. Application of MCP-PAB conditions to epithelial ALI cultures caused marked acute structural damage, including disruption of adherens and tight junctions, increased permeability and induction of stress responses, as shown by enrichment of the UPR genes and modulation of the alarmin IL-33 and its receptor IL1RL1. These in vitro findings were supported by observations in bronchial biopsies and by global gene expression analysis of epithelial brushings from severe asthmatics without or with GORD and healthy controls.

GORD is a chronic disorder caused by abnormal reflux of acid, pepsin and bile acids, defined as time of acid exposure >6% during 24-hr monitoring of oesophageal pH [START_REF] Roman | Ambulatory reflux monitoring for diagnosis of gastro-esophageal reflux disease: Update of the Porto consensus and recommendations from an international consensus group[END_REF]. Combined with impedance measurement, pH monitoring allows the detection of acidic (pH<4), weakly acidic (pH 4-7) and non-acid reflux (pH>7), the latter occasionally persisting despite treatment with PPI [START_REF] Pauwels | The effect of gastric juice on interleukin-8 production by cystic fibrosis primary bronchial epithelial cells[END_REF]. Whether and to what extent gastric juice contents penetrate the lungs in subjects with GORD has been uncertain [START_REF] Hunt | The Potential Role of Aspiration in the Asthmatic Airway[END_REF], although our own studies have provided evidence that clinical GORD is associated with changes in several biomarkers (11,[START_REF] Perotin | Epithelial dysregulation in obese severe asthmatics with gastro-oesophageal reflux[END_REF]. When deciding on the composition of the ingredients in the MCP-PAB for in vitro testing, we took into account physiological concentrations in gastric secretions of acid [START_REF] Mertens | Gastric juice from patients "on" acid suppressive therapy can still provoke a significant inflammatory reaction by human bronchial epithelial cells[END_REF], pepsin [START_REF] Roberts | Secretion of total pepsin and pepsin 1 in healthy volunteers in response to pentagastrin and to insulin-induced hypoglycaemia[END_REF] and total bile acids [START_REF] Farré | Short exposure of oesophageal mucosa to bile acids, both in acidic and weakly acidic conditions, can impair mucosal integrity and provoke dilated intercellular spaces[END_REF] and previous reports of effects of pepsin ( 9) and bile acids [START_REF] Perng | Bile acids induce CCN2 production through p38 MAP kinase activation in human bronchial epithelial cells: a factor contributing to airway fibrosis[END_REF] on epithelial cells. Based on dose-ranging experiments, we chose 50µg/ml of pepsin and 250µM chenodeoxycholic acid (CDC) at pH 5 for 30 min because this resulted in measurable damage without causing extreme cytotoxicity. Thus, we observed enlargement of intercellular spaces, disruption of intercellular junctions and increased permeability, effects similar to observations in vivo in oesophageal and laryngeal epithelium exposed to chronic refluxate [START_REF] Franchi | Dilation of intercellular spaces is associated with laryngo-pharyngeal reflux: an ultrastructural morphometric analysis of laryngeal epithelium[END_REF][START_REF] Bor | Reflux esophagitis, functional and non-functional[END_REF]. This, coupled with previous studies showing that ALI cultures derived from severe asthmatic donors exhibit phenotypic features similar to those found in vivo [START_REF] Xiao | Defective epithelial barrier function in asthma[END_REF][START_REF] Gras | An ex vivo model of severe asthma using reconstituted human bronchial epithelium[END_REF], led us to conclude that exposure of ALI cultures to MCP-PAB conditions is a reliable model to analyse the effect of reflux on the bronchial epithelium in severe asthma.

Our study revealed a marked reflux-induced increase in the nuclear expression of the alarmin IL-33, as well as upregulation of IL1RL1, the gene encoding the IL-33 receptor. IL-33 is a member of the IL-1 cytokine family localized in the nucleus of airway epithelial cells and its release can be triggered by damage caused, for example, by allergens or viruses [START_REF] Jackson | IL-33dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo[END_REF][START_REF] Wang | Bronchial Allergen Challenge of Patients with Atopic Asthma Triggers an Alarmin (IL-33, TSLP, and IL-25) Response in the Airways Epithelium and Submucosa[END_REF]. It is a known asthma susceptibility gene [START_REF] Moffatt | A large-scale, consortium-based genomewide association study of asthma[END_REF][START_REF] Torgerson | Metaanalysis of genome-wide association studies of asthma in ethnically diverse North American populations[END_REF] and plays a crucial role in type-2 innate immunity through activation of group 2 innate lymphoid cells (ILC-2) to trigger production of IL-4, IL-5, IL-13 [START_REF] Chan | IL33: Roles in Allergic Inflammation and Therapeutic Perspectives[END_REF]. Our findings of IL-33 upregulation in bronchial biopsies from severe asthma with GORD are in concordance with the observed upregulation of IL-33 nuclear expression in the oesophageal mucosa in patients with reflux oesophagitis [START_REF] Shan | Epithelial-derived nuclear IL-33 aggravates inflammation in the pathogenesis of reflux esophagitis[END_REF] and symptoms of heartburn [START_REF] Sei | Esophageal Epithelial-Derived IL-33 Is Upregulated in Patients with Heartburn[END_REF].

Refluxate-induced damage also included a response to oxidative stress through PERK-mediated UPR [START_REF] Fribley | Regulation of apoptosis by the unfolded protein response[END_REF]. A recent study, using an oesophageal squamous epithelial cell line, identified bile acid-mediated activation of the PERK-mediated UPR [START_REF] Sharma | Unconjugated secondary bile acids activate the unfolded protein response and induce golgi fragmentation via a src-kinase-dependant mechanism[END_REF]. Our study provides the first evidence of refluxate-triggered UPR activation in the airway epithelium. PERK is a type I endoplasmic reticulum (ER) transmembrane protein activated by misfolded proteins inside the ER. Its stimulation induces transcription of UPR related genes, leading to autophagy, apoptosis and redox homeostasis [START_REF] Pathinayake | Understanding the Unfolded Protein Response in the Pathogenesis of Asthma[END_REF]. UPR is considered a master regulator in inflammatory diseases and its role in asthma development has been suggested [START_REF] Pathinayake | Understanding the Unfolded Protein Response in the Pathogenesis of Asthma[END_REF]. UPR can be activated by various asthma triggers, including allergens, cigarette smoke and viruses, and regulates oxidative stress in asthma [START_REF] Pathinayake | Understanding the Unfolded Protein Response in the Pathogenesis of Asthma[END_REF]. UPR regulates NKκB activity and NFκB-mediated inflammation and can induce apoptosis in case of prolonged activation of ER stress. The relatively limited enrichment in the PERK-mediated UPR process that we observed in exposed cells from severe asthmatics may reflect an ineffective response to multiple types of damage and so explains the vulnerability of the bronchial epithelium in severe asthma to a range of environmental challenges.

We observed MCP-PAB-induced epithelial changes in cultures from both normal and severe asthma donors. However, MCP-PAB had a more pronounced impact on the structural and functional properties of the asthmatic epithelium, including a greater increase in epithelial permeability and a higher number of MCP-PAB-associated DEGs. While our ex vivo transcriptome analysis of bronchial brushings did not completely match our in vitro results from ALI cultures, this may be because the in vitro model represents a single acute stress event caused by exposure to MCP-PAB conditions which do not fully reflect the complexity of gastric juice, whereas the clinical condition of GORD is characterized by repeated exposures to various components of gastric juice. In addition, severe asthma patients with GORD treated with PPI may present a dysregulated aerodigestive microbiome, with a potential role in severe asthma [START_REF] Hufnagl | Dysbiosis of the gut and lung microbiome has a role in asthma[END_REF][START_REF] Wang | Altered gut microbiome compositions are associated with the severity of asthma[END_REF]. Nonetheless, we were able to identify 15 dysregulated genes in brushings from severe asthmatics with GORD among the 37 top dysregulated genes in MCP-PAB-exposed ALI cultures, with the extent of dysregulation being higher in cultures from severe asthmatics than in those from healthy controls. Amongst the DEGs were genes involved in oxidative stress responses (CHAC1, BACH2), cell adhesion (CEACAM1), cytoskeleton organization (CDC42EP1) and cilia formation (GMNC), pointing to impact on epithelial structure regulation.

Exposure of severe asthma cultures to refluxate also caused enrichment in EGFR-and cell migration-related processes that were not changed in cultures from healthy individuals. Our results suggest that refluxate might contribute to defective epithelial barrier function and EGFRmediated remodelling, key features of asthma [START_REF] Xiao | Defective epithelial barrier function in asthma[END_REF][START_REF] Cras | Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma[END_REF].

In summary, our study has identified a direct impact of refluxate on the airway epithelial structure, barrier permeability and modulation of gene expression, including UPR, responses to oxidative stress and wound healing processes. This suggests a possible role for GORD in increasing exposure of the subepithelial airway mucosa to allergens and infectious pathogens, resulting in increased risk of inflammation and exacerbations, as well as a possible role in airway remodelling, a key feature of severe asthma. These results suggest the need for research into alternative therapeutic management of GORD in severe asthma. E2. Epithelial cells were harvested by bronchoscopic brushings and samples were either processed into RNAlater for subsequent RNAseq analysis or used in primary bronchial epithelial cells (PBEC) culture.

Bronchial biopsies were also taken and embedded into a glycomethacrylate resin for immunohistochemical analyses.

Development of the refluxate mix for use in epithelial culture

Prior to cultures with epithelial cells obtained from patients and controls, dose-ranging and time-course studies were conducted with 16HBE cell line cells using varying compositions of multiple challenge protocol (MCP) containing pepsin (50 to 150 µg/ml; Sigma-Aldrich, St Louis, MO, USA), hydrochloric acid (pH 1.5 to 5) and chenodeoxycholic acid (CDC) (50 µM to 5 mM; Sigma-Aldrich, St Louis, MO, USA). Cells were maintained in minimum essential medium (MEM) with Glutamax and F12 and supplemented with 10% foetal bovine serum (FBS) and penicillin/streptomycin (Life technologies, Paisley, UK) on PureCol collagen I (Advanced BioMatrix, San Diego, CA, USA) coated culture flasks.

Experiments were carried out using collagen-coated Transwell® permeable supports (diameter 6.5 mm, polyester membrane with 3 µm pores, Corning Life Sciences, Amsterdam, The Netherlands). Cells were seeded at a density of 0.7x10 5 cells in 200 µL of growth medium; the basolateral compartment contained 500 µL of the same medium. Medium exchange was carried out every 2-3 days. In culture, the 16HBE cells formed a polarized epithelial sheet within seven days, as monitored by measuring the transepithelial resistance (TER). Cells with a TER >700 Ω/cm 2 on day 7 were used for experiments.

Culture media containing varying concentrations of hydrochloric acid, pepsin and/or chenodeoxycholic acid were applied to the apical pole of cells for 5 to 30 minutes. Apical supernatants were collected and centrifuged (10.000 rpm 10 min at 4°C) for assessment of cytotoxicity. The epithelial cells were then washed twice and fresh control culture media was replaced. After 4hr of recovery, micro and macromolecular permeability were measured by TER and FITC dextran 4kDa respectively. The cells were then lysed using Trizol lysis reagent (Life Technologies, Paisley, UK) and frozen at -80°C until analysed.

Exposure of 16HBE cells to hydrochloric acid was associated with dose-dependent damage of the epithelium (Figure E1). Comparison between timepoints showed no significant differences in signals (data not shown). Pepsin did not have any additional impact on cell viability at different concentrations (50 to 150µg/ml) or pH (1.5, 2.5 and 5) after 30 minutes of exposure. Chenodeoxycholic acid exposure induced dose-dependent cytotoxicity, with no additional impact of CDC 500 µM at pH5. We, therefore, defined pH5 + pepsin 50µg/ml + CDC 500µM as the working multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) at this stage of culture optimization and chose 30 minutes as the time of exposure. This MCP-PAB model induced a mean 3.5% cytotoxicity in 16HBE cells and a mean 39% decrease in ionic epithelial permeability.

Primary epithelial cultures

Having optimised the culture and AR exposure conditions for 16HBE cells, we went on to optimise the same for primary bronchial epithelial cells (PBEC). PBEC were obtained from healthy and asthmatic donors by fiberoptic bronchoscopy. After adding RPMI, 10% FBS and 2% penicillin/streptomycin, cells were centrifuged at 1200rpm for 5min at room temperature. The cell pellets were re-suspended in Airway Epithelial Cell Growth Medium (Promocell, Heidelberg, Germany) and seeded into PureCol collagen I coated culture flasks. After achieving confluence, the cells were transferred into Transwell® permeable supports and cultured at air-liquid interface (ALI). After 21 days, the epithelial cells formed a differentiated pseudostratified epithelium. Using trans-epithelial resistance (TER) as a measure of differentiation, cultures with a TER>1000 were deemed fully differentiated and were, therefore, selected for AR experiments. The ALI cultures were starved overnight prior to start of the exposure. In these conditions CDC 500µM was found to be cytotoxic; therefore, its concentration was reduced to 250µM.

The final AR was therefore defined as pH5 + pepsin 50µg/ml + CDC 250µM and this was applied for 30 min.

Analysis of the effect of multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) on epithelial cultures

Assessment of cytotoxicity

Lactate dehydrogenase (LDH) release was measured using a CytoTox 96 Nonradioactive Cytotoxicity Assay (Promega, Southampton, UK), according to the manufacturer's instructions. All samples were assayed in duplicate. Intracellular LDH in control cultures was determined by lysing cells with 1%

Triton X-100 in Airway Epithelial Cell Growth Medium. LDH release in each well was calculated as a percentage of total LDH, and values for control wells were subtracted from challenged wells to give the percentage of total LDH released as a result of challenge.

Measurement of epithelial permeability

Transepithelial electrical resistance (TER) was measured using an EVOM voltohmmeter (World Precision Instruments, Hitchin, UK). Fluorescein isothiocyanate (FITC)-dextran 4 kDa (Sigma-Aldrich, Poole, United Kingdom) was applied to cells apically and incubated for 4 hours at 37°C. Basolateral dextran passage was analyzed with a Fluoroskan Ascent FL2.5 reader (Thermo Fisher, London, United Kingdom).

Immunostaining and electron microscopy

Immunostaining ALI cultures were analysed using immunofluorescence. ALI cultures were fixed with 4% paraformaldehyde and then permeabilized with 0.1% triton X 100 for 15 min at room temperature and blocked with BSA (1% BSA, 0.1% Tween 20 in PBS) for 1 hr. Cells were stained with mouse monoclonal anti-ZO-1 (1/100; BD biosciences, San Jose, CA, USA), mouse monoclonal anti-E-cadherin Bronchial biopsies were analysed using immunohistochemistry. Bronchial biopsies were processed as previously described ( 3) and embedded into paraffin; 4-µm sections were cut from all suitable biopsies and stained by immunohistochemistry using goat polyclonal anti-human IL-33 (R&S Systems, Abingdon, UK). Epithelial cells were counted and results expressed as positive cells per total epithelial cells.

Electron Microscopy

PBEC in transwells were fixed in 3% glutaraldehyde, 4% formaldehyde, 0.1M PIPES buffer pH7.2 for 30 min at RT, then rinsed with 0.1M PIPES buffer pH7.2 and subsequently post-fixative 1% Osmium tetroxide 0.1M PIPES buffer pH7.2. Transwells were stained enbloc in 2% Uranyl Acetate then dehydrated through a graded series of ethanols into acetonitrile and into embedded in Spurr resin.

Ultrathin sections (90nm) were cut and stained with Reynolds lead citrate. Sections were analysed using a Hitachi HT7700 transmission electron microscope.

Cytokine analysis

Interleukin 8 (IL-8) concentrations in culture media from the apical and basolateral chambers were measured using an IL-8 DuoSet ELISA (R&D, Abingdon, UK) in accordance with manufacturer's instructions. Each sample was evaluated using 2 technical replicates and the mean value was used for subsequent statistical analysis. IL-6, TNFα, IL1α were measured using VPlex (MSD, Maryland, USA) according to manufacturer's recommendations.

Analysis of gene expression in epithelial brushings and differentiated cells

RNA extraction

Total RNA was extracted from primary bronchial epithelial samples using the Qiagen RNeasy kit. RNA quality and concentration were measured using an RNA Nano chip on the Agilent Bioanalyser 2100.

Samples with total RNA concentration ≥50ng/µl, RIN ≥6.8 and OD 260/280 were taken for cDNA library preparation and sequencing.

cDNA library preparation and sequencing cDNA libraries were prepared using NEBNext Ultra (non-stranded) mRNA library prep kit with polyA pulldown for mRNA enrichment (Novogene Inc). Library quality was assessed using a broad range DNA chip on the Agilent Bioanalyser 2100. Library concentration was assessed using Qubit and q-PCR.

Libraries were pooled. Paired-end 150bp sequencing to a depth of 20M reads (epithelial brushings) or 50M reads (differentiated cells) was performed on an Illumina HiSeq2500 by Novogene Inc (Cambridge, UK).

Data processing

Quality control

Raw FASTQ reads were subjected to adapter trimming and conservative quality filtering (reads containing N > 10%, reads where >50% of read has Qscore<= 5) by Novogene Inc. Quality of sequence was assessed using fastqc, aggregated using multiqc.

Alignment

Paired FASTQ files were aligned to human genome build 38 using gencode v29 gene annotations and STAR v2.

Differential gene expression analysis

Count data from HTseq was subjected to quantile filtration to remove genes with more than 25% of samples with a read count of 0. Count data was normalised by library size to calculate counts per million (CPM). CPM data was normalised by distribution using the trimmed Mean of the M-values (TMM) approach using edgeR (7,8).

Gene ontology was performed by AmiGO (9-11) using the top 300 upregulated differentially expressed genes (DEGs) in the group of severe asthmatic with GORD compared to the group of severe asthmatics with no GORD.

qRT-PCR

RNA was reverse transcribed to cDNA using a Precision Reverse Transcription kit (PrimerDesign, Southampton, UK) according to the manufacturer's instructions. Expression of CXCL8 (Applied Biosystems, Paisley, UK) and IL-33 (Primer Design, Chandler's Ford, UK) was determined using probebased qPCR, whereas expression of the housekeeping genes ubiquitin C and glyceraldehyde 3phosphate dehydrogenase was determined using a probe-based duplex primer mix (PrimerDesign). Fold change in gene expression relative to time-matched controls was determined using the ΔΔCt method.

SUPPLEMENTARY TABLES

Figure 1 .

 1 Figure 1. Effects of Multiple Challenge Protocol using pepsin, acid pH and bile acid on Epithelial Permeability. Bronchial epithelial ALI cultures from healthy controls (HC) (N=5) and severe asthmatic (SA) (N=8) donors were untreated or exposed to multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) for 30 mins, washed and allowed to recover for 4 hours before ionic (a) and macromolecular (b) permeability were measured. * p<0.05 vs control; ** p<0.005 vs control.

Figure 2 .

 2 Figure 2. Stimulation of Epithelial Cytokine Release by Multiple Challenge Protocol using pepsin, acid pH and bile acid . Bronchial epithelial ALI cultures from healthy controls (HC) (N=4) and severe asthmatic (SA) (N=6) donors were untreated or exposed to Multiple Challenge Protocol using pepsin, acid pH and bile acid (MCP-PAB) for 30 mins, washed and allowed to recover for 4 hours before CXCL8 (A), IL-1α (B), TNFα (C) and IL-6 (D) protein release was measured in apical secretions. *p<0.05 vs control. ** p<0.005 vs control.

Figure 3 .

 3 Figure 3. Changes in Epithelial Gene Expression caused by Multiple Challenge Protocol using pepsin, acid pH and bile acid in vitro. Bronchial epithelial ALI cultures from healthy controls (HC) (N=5) and severe asthmatic (SA) (N=6) donors were untreated or exposed to multiple challenge protocol using pepsin, acid pH and bile acid for 30 mins, washed and allowed to recover for 4 hours before harvesting for RNA-seq. Heatmap of the top dysregulated epithelial genes from low (blue) to high (red) levels of expression. For exact fold-change, see TableE2.

Figure 4 .

 4 Figure 4. Regulation of Epithelial Expression of IL-33 by Multiple Challenge Protocol using pepsin, acid pH and bile acid . Bronchial epithelial ALI cultures from severe asthmatic (SA) donors were untreated or exposed to multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) for 30 mins, washed and allowed to recover for 4 hours before fixing and immunofluorescence staining. Images show IL-33 nuclear staining (green) and 4′,6-diamidino-2-phenylindole (DAPI) (red). Images are representative of experiments using ALI cultures from 6 donors. Scale bar = 25µm.

Figure 5 .

 5 Figure 5. Epithelial IL-33 Expression is Increased Severe Asthma with GORD. Immunohistochemical staining for IL-33 in bronchial biopsies from healthy control participants without GORD (HC), severe asthmatics with no documented GORD (SA-no GORD; n=5), severe asthmatics with documented GORD who had abstained from their regular PPI treatment for 2 weeks (SA-GORD; n=4). Panel A shows typical patterns of IL-33 immunostaining and panel B shows quantitation of positive nuclei expressed as percentage of total epithelial cells.

( 1 /

 1 250; Cell Signaling, London, UK) or goat polyclonal anti-human IL-33 (1/20; R and D Systems, Abingdon, UK) diluted in 1% BSA, 0.1% Tween 20 in PBS overnight at 4°C. Cultures were then washed with 0.1% Tween 20 in PBS 4 times and incubated for 2 hrs with donkey anti-goat Alexa fluor 488 labelled secondary antibody (Life Technologies, Carlsbad, CA, USA) or goat anti-mouse Alexa fluor 647 labelled secondary antibody (Biolegend, San Diego, CA, USA). Cell nuclei were counterstained with DAPI. Images were acquired using a Leica SP8 laser-scanning confocal microscope (Leica Microsystems, UK).

Figure E1 .

 E1 Figure E1. Development of the refluxate mix. Exposure of 16HBE cell cultures to hydrochloric acid (A, B), chenodeoxycholic acid (C, D) and/or artificial refluxate (E, F) induced cytotoxicity (A, C, E) and increase in epithelial ionic permeability (B, D, F). CDC: Chenodeoxycholic Acid (µM); MCP-PAB: multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) * p<0.05; ** p<0.005; ***p<0.0005

Figure E3 .

 E3 Figure E3. Disruption of Epithelial Tight junctions by Artificial Refluxate. Bronchial epithelial ALI cultures from severe asthmatic donors were untreated or exposed to multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) for 30 mins, washed and allowed to recover for 4 hours before fixing and immunofluorescence staining. Panel A shows ZO-1 (green) and 4′,6-diamidino-2phenylindole (DAPI) (red) and Panel B shows E-cadherin (green) and 4′,6-diamidino-2-phenylindole (DAPI) (red). Images are representative of experiments using 6 donors. Scale bar = 25µm.

Figure E4 .

 E4 Figure E4. Stimulation of Epithelial Cytokine expression by artificial refluxate. Bronchial epithelial ALI cultures from healthy controls (HC) (N=5) and severe asthmatic (SA) (N=6) donors were untreated (CTL) or exposed to multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) for 30 mins, washed and allowed to recover for 4 hours before RNA extraction for CXCL8 (A) and IL-1α (B) gene expression analysis. * p<0.05 vs control. ** p<0.005 vs control.

Table 1 .

 1 Characteristics of participants

	Tables
		Healthy controls	Severe asthmatics	p value	SA-no GORD	SA-GORD	SA-GORD+PPI
	n	15	18		4	6	8
	Age, yrs	41.5 ± 12.9	55.7 ± 8.1	0.0005	52.5 ± 6.2	54.3 ± 9.2	58.25 ± 7.99
	Age of asthma onset, yrs		23.7 ± 21.0		24.0 ± 25.7	27.3 ± 23.6	20.75 ± 19.40
	Gender (F/M)	11/4	11/7	0.48	3/1	6/0	2/6
	Never smoker	11	8	0.28	3	4	7
	BMI, kg/m²	25.76 ± 3.68	32.55 ± 6.74	0.001	30.28 ± 5.97	37.0 ± 5.5	30.4 ± 6.9
	Obese (BMI>30)	1	10	0.004	2	5	3
	Diagnosed GORD	0	14	<0.0001	0	6	8
	Atopy, n	4	11	0.08	1	4	6
	Sputum
	Eosinophils, % total cells	0.14 [0-0.51] 1.01 [0.27-3.57]	0.10	1.97 [0.26-4.83]	0.51 [0.13-5.23]	1.13 [0.34-2.95]
	Neutrophils, % total cells 44.3 [20.1-74.7] 64.1 [37.8-73.1]	0.44	52.9 [31.4-68.3]	65.0[60.3-73.4]	63.4 [11.7-75.3]
	FEV1, % predicted	116.6 ± 23.4	89.7 ± 23.6	0.004	82.5 ± 23.0	105.8 ± 24.4	78.3 ± 15.7
	FVC, % predicted	121.7 ± 19.6	110.7 ± 20.0	0.14	99.4 ± 12.1	124.8 ± 25.0	104.1 ± 9.4
	FEV1/FVC	0.80 ± 0.04	0.66 ± 0.12	0.0001	0.69 ± 0.12	0.72 ± 0.08	59.2 ± 12.2
	FeNO		24.2 ± 11.1		33.0 ± 17.0	28.8 ± 11.0	19.8 ± 8.8
	Exacerbations (last year)		4.1 ± 3.4		3.3 ± 1.5	3.7 ± 2.0	4.7 ± 4.9
	ACQ6		12.8 ± 6.5		6.5 ± 4.8	15.5 ± 6.2	14 ± 5.88
	SGRQ
	Activity score	5.16 ± 6.55	58.12 ± 24.52 <0.0001	43.89 ± 12.02 62.10 ± 23.10 72.94 ± 16.66
	Impact score	0.32 ± 1.15	36.47 ± 15.82 <0.0001	20.45 ± 10.08 39.10 ± 18.28 41.75 ± 12.94
	Symptoms score	8.44 ± 8.93	65.13 ± 22.81 <0.0001	55.51 ± 23.90 65.28 ± 25.56 75.73 ± 13.83
	Total score	3.16 ± 2.42	47.79 ± 17.01 <0.0001	30.05 ± 12.85 50.42 ± 17.20 56.84 ± 13.83
	Hull Cough questionnaire	1.0 ± 2.0	36.05 ± 21.97 <0.0001	23.75 ± 13.23 32.50 ± 20.54 34.43 ± 13.10
	Treatment
	Proton pump inhibitor, n	0	8	0.0036	0	0	8
	Results are expressed as numbers, mean ± SD or median [IQ25-75]. No significant difference within
	the groups of severe asthma patients

Table 2 .

 2 Top upregulated biological processes in multiple challenge protocol exposed cultures when compared with control cultures (details provided if fold enrichment >5, p-value<0.05)

	HC	SA

Table 3 .

 3 Differential epithelial expression of 37 genes in ALI cultures and expression of these genes in bronchial brushings. Foldchanges are coloured from blue (downregulation) to red (upregulation): colour intensity reflects magnitude.

			ALI cultures				Bronchial brushings
		SA MCP vs HC CTL	SA MCP vs SA CTL	SA-GORD vs Health	SA-GORD vs SA-no GORD	SA-no GORD vs Health
		fold change p value	fold change p value	fold change	p value	fold change	p value	fold change	p value
	IL1RL1	14.22	0.0234	11.84	0.014
	CYP26A1	10.31	0.0102	2.26	0.0226	-0.38	0.2119	-0.59	0.015	0.4881 0.2138
	CHAC1	6.38	0.0000	8.53	0.0000	0.57	0.0014	0.4	0.078	0.1204 0.3519
	PLAUR	5.06	0.0036	5.71	0.0013	0.78	0.1908	0.22	0.7903	0.4588 0.3107
	BCL2A1	4.48	0.0174	7.26	0.006	1.19	0.2114	0.42	0.7148	0.5371 0.3406
	LIF	4.39	0.0345	3.49	0.0271	0.03	0.8773	0.28	0.4522	-0.1967 0.2768
	ASNS	4.37	0.0000	3.77	0.0000
	EHD3	4.16	0.0001	2.5	0.0001	0.43	0.0314	-0.11	0.6064	0.6105 0.0122
	DUSP5	4.13	0.001	4.72	0.0003	0.04	0.8965	0.05	0.9213	-0.0097 0.9756
	FOSL1	3.89	0.0011	3.6	0.0005
	AKAP12	3.59	0.0000	2.43	0.0000	0.62	0.0429	0.5	0.2501	0.0834 0.7275
	EMP1	3.46	0.0001	3.91	0.0000	0.57	0.0126	-0.32	0.272	1.314	0.008
	IL36G	3.33	0.0196	5.99	0.0052
	IL1RN	2.83	0.0003	2.15	0.0002	1.54	0.048	0.04	0.9582	1.4514	0.073
	BACH2	2.61	0.0001	3.84	0.0000	0.79	0.0242	0.45	0.3752	0.2311 0.3876
	CEACAM1	2.55	0.0001	2.14	0.0000	1.05	0.0031	0.77	0.1259	0.1556 0.3661
	SEMA7A	2.51	0.0068	3.36	0.0011	0.86	0.0559	0.67	0.2766	0.1151 0.7938
	SERPINB9	2.28	0.0586	5.33	0.0151	0.47	0.1701	0.1	0.827	0.3359 0.4284
	MAFF	2.08	0.0002	2.18	0.0001	0.1	0.7534	0.38	0.4754	-0.2083 0.4484
	LIPH	1.88	0.0001	2.12	0.0000	-0.22	0.046	0.17	0.5231	-0.3343 0.0044
	TICAM1	1.81	0.0001	1.7	0.0001	0.7	0.0171	0.36	0.3158	0.2518 0.2377
	HERPUD1	1.72	0.0009	1.65	0.0003	0.17	0.0537	-0.12	0.3178	0.3201	0.007
	DDIT3	1.65	0.0096	1.92	0.0023	0.35	0.037	-0.16	0.335	0.6075 0.0004
	CDC42EP1	1.52	0.0000	1.49	0.0001	1.63	0.029	1.91	0.1866	-0.0962 0.7368
	SESN2	1.42	0.0000	1	0.0000	0.21	0.1479	-0.11	0.517	0.3598 0.0141
	SLC7A11	1.41	0.0002	2.13	0.0000	0.25	0.4279	1.25	0.0364	-0.4458 0.2045
	LPIN1	1.38	0.0000	1.61	0.0000	-0.01	0.895	0.19	0.2361	-0.1672 0.0609
	ERRFI1	1.32	0.0000	1.9	0.0000	-0.03	0.7968	0.12	0.5169	-0.1333 0.1547
	GRPEL2	1.15	0.0009	1.42	0.0002	-0.13	0.3447	0.06	0.7801	-0.185 0.2209
	HSPA5	1.12	0.0000	1.62	0.0000	-0.05	0.7581	0.1	0.6946	-0.1369 0.3707
	NCOA7	0.59	0.0073	0.85	0.0001	-0.35	0.0361	0.34	0.1864	-0.5129 0.0139
	ODC1	0.51	0.0119	1.22	0.0001	-0.25	0.0021	-0.2	0.003	-0.0647 0.4584
	OCA2	-0.28	0.2787	-0.05	0.8474	0.84	0.0171	-0.37	0.3526	1.9081 0.0177
	CYP1B1	-0.32	0.3627	-0.33	0.4407
	CYP1A1	-0.55	0.0343	-0.51	0.1031
	SOX2	-0.7	0.0001	-0.67	0.0000	-0.2	0.038	-0.05	0.7193	-0.1535	0.047
	GMNC	-0.81	0.0000	-0.72	0.0047	-0.58	0.0002	-0.14	0.6958	-0.5164 0.0075
	MCP: multiple challenge protocol ; CTL: Control; HC: healthy control; SA-no GORD: severe
	asthmatics with no documented GORD; SA-GORD: severe asthmatics with documented GORD who had
	abstained from their regular PPI treatment for 2 weeks.

Table E1 .

 E1 Top DEGs fold changes relative to control in healthy subjects (HC) and severe asthmatics (SA) ALI cultures exposed to multiple challenge protocol using pepsin, acid pH and bile acid (MCP-PAB) from negative (blue) to increasingly positive (increasing intensity of red).

		HC	SA Gene name
	IL1RL1		11,8 Interleukin 1 Receptor Like 1
	CHAC1	8,9	8,5 ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1
	IL36G	2,9	6	Interleukin 36 Gamma
	PLAUR	2,5	5,7 Plasminogen Activator, Urokinase Receptor
	SERPINB9		5,3 Serpin Family B Member 9
	DUSP5	2,6	4,7 Dual Specificity Phosphatase 5
	EMP1	1,3	3,9 Epithelial Membrane Protein 1
	BACH2		3,8 BTB Domain And CNC Homolog 2
	FOSL1		3,6 FOS Like 1, AP-1 Transcription Factor Subunit
	SEMA7A		3,4 Semaphorin 7A (John Milton Hagen Blood Group)
	EHD3		2,5 EH Domain Containing 3
	AKAP12		2,4 A-Kinase Anchoring Protein 12
	IL1RN		2,2 Interleukin 1 Receptor Antagonist
	CEACAM1		2,1 CEA Cell Adhesion Molecule 1
	SLC7A11		2,1 Solute Carrier Family 7 Member 11
	LIPH	1,3	2,1 Lipase H
	ERRFI1	1,6	1,9 ERBB Receptor Feedback Inhibitor 1
	HSPA5	1	1,6 Heat Shock Protein Family A (Hsp70) Member 5
	LPIN1		1,6 Lipin 1
	SOX2		-0,7 SRY-Box Transcription Factor 2
	CYP26A1	11,7		Cytochrome P450 Family 26 Subfamily A Member 1
	ASNS	6,6		Asparagine Synthetase (Glutamine-Hydrolyzing)
	LIF	3,9		LIF Interleukin 6 Family Cytokine
	BCL2A1	3,8		BCL2 Related Protein A1
	DDIT3	2,7		DNA Damage Inducible Transcript 3
	MAFF	2		MAF BZIP Transcription Factor F
	HERPUD1	1,6		Homocysteine Inducible ER Protein With Ubiquitin Like Domain 1
	SESN2	1,5		Sestrin 2
	NCOA7	1,5		Nuclear Receptor Coactivator 7
	CDC42EP1	1,4		CDC42 Effector Protein 1
	TICAM1	1,4		Toll Like Receptor Adaptor Molecule 1
	GMNC	-0,7		Geminin Coiled-Coil Domain Containing

JMP acknowledges the support of the European Respiratory Society (Fellowship LTRF 2017) and of the Asthma, Allergy and Inflammation Research Charity. JMP, GW, KT, AA, RAR, JAW, CB, DED have nothing to disclose. JPRS reports being Director and shareholder in TopMD Precision Medicine Ltd. PH reports personal fees from GSK outside the submitted work. RD reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as member of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline.


Angélique Gougelet
email: angelique.gougelet@inserm.fr

Chiara Sartor

Laura Bachelot

Cécile Godard

Carmen Marchiol

Gilles Renault

Frédéric Tores

Patrick Nitschke

Catherine Cavard

Benoit Terris

Christine Perret

Sabine Colnot

Antitumour activity of an inhibitor of miR-34a in liver cancer with β-catenin-mutations

Keywords: hepatocellular carcinoma, β-catenin, miR-34a, HNF-4α, miRNA-based therapies

come

INTRODUCTION

Liver cancer is the third leading cause of cancer-related death worldwide.

Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer, which mostly affects men with cirrhosis. HCC frequently appears in a context of chronic liver disease caused by infection with the hepatitis B and C viruses (HBV and HCV, respectively), alcohol consumption, obesity or genotoxic exposure ( [START_REF] Knudsen | The changing landscape of hepatocellular carcinoma: etiology, genetics, and therapy[END_REF] for a review). We, and others, have shown that mutations of the CTNNB1 gene, encoding β-catenin, are observed in 15 to 40% of HCCs [START_REF] De | Somatic mutations of the betacatenin gene are frequent in mouse and human hepatocellular carcinomas[END_REF][START_REF] Miyoshi | Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3[END_REF]. HCCs with β-catenin mutation constitute one of the two subclasses of HCC [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF], and are thought to follow a particular pattern of pathogenesis, as most are well differentiated, cholestatic and with a better prognosis [START_REF] Audard | Cholestasis is a marker for hepatocellular carcinomas displaying beta-catenin mutations[END_REF]. Several years ago, we engineered a mouse model in which β-catenin signaling was overactivated exclusively in the liver, by the tamoxifeninducible and Cre-Lox-mediated inactivation of the Apc (Apc KO ) gene, encoding a tumor suppressor, which is an inhibitor of the Wnt/β-catenin pathway, and the liver "zonation-keeper" [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF][START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. In this model, the absence of Apc in single hepatocytes results in the development of β-catenin-activated liver tumors with properties similar to those observed in human patients [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. If Apc is deleted in all hepatocytes, the βcatenin signaling in hepatocytes leads to the induction of a transcriptional program very similar to that of human HCC with β-catenin mutation [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF][START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF].

The molecular pathogenesis of HCC has been elucidated [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF], but the incidence and mortality of HCC remain high, despite the use of the tyrosine kinase inhibitor sorafenib. Different therapeutic strategies could therefore be used to target tumors in accordance with their pathogenic pathways. MicroRNAs (miRNAs) have recently emerged as potent diagnostic and prognostic biomarkers, and numbers of reports devoted to miRNAs have shown that these molecules act as oncogenes or tumor suppressors [START_REF] Babashah | The oncogenic and tumour suppressive roles of microRNAs in cancer and apoptosis[END_REF]. The global analysis of the miRNAs expressed in liver tumors highlighted a miRNA signature common to all types of HCC [START_REF] Gougelet | microRNA: new diagnostic and therapeutic tools in liver disease?[END_REF][START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF][START_REF] Murakami | Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues[END_REF]. A loss of miR-122 [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF], miR-29 [START_REF] Xiong | Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma[END_REF], and miR-124 [START_REF] Furuta | miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma[END_REF] has been described. Inversely, some oncogenic miRNAs have been identified: miR-21 [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF], miR-221/miR-222 [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF], and miR-224 [START_REF] Wang | Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target[END_REF]. Despite the number of global analyses, the role of miR-34a in liver tumorigenesis remains unclear. Even if this miRNA is currently described as a tumor suppressor in various cancers [START_REF] Lodygin | Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer[END_REF], two studies suggested that miR-34a levels decrease in HCCs, mainly HBV+ [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF][START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF], while other studies reported an induction of miR-34a, particularly in HCC with β-catenin mutations [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF][START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF].

We aimed to identify candidate microRNAs, regulated by β-catenin, potentially involved in liver tumorigenesis. We observed that miR-34a was overexpressed in response to β-catenin overactivation; the levels of miR-34a being inversely correlated with those of HNF-4α (hepatocyte nuclear receptor α), another key factor for hepatocyte differentiation, which we have shown to antagonize β-catenin function [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF].

MiR-34a induction was observed specifically in mouse and human liver tumors with β-catenin mutations, and miR-34a was found to play an oncogenic role in the liver.

These findings call into question the systematic use of miR-34a mimics to combat liver tumor growth (http://www.mirnarx.com/pipeline/mirna-MRX34.html). Instead, we provide here proof-of-concept that a locked nucleic acid (LNA)-based inhibitor of miR-34a may be useful for treatment, exclusively in HCCs with β-catenin mutation.

METHODS

Animals and reagents

Transgenic mice, liver sampling and immunostaining have been described elsewhere [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF][START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF][START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF], and detailed in the supporting materials.

Hepatocyte isolation

To directly lyse hepatocytes after collagenase perfusion, i.e. for RNA or protein extraction, the transgenic livers were perfused six days after tamoxifen injection (1.5 mg) as previously described [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF][START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF]. Non-parenchymal cells were excluded in the supernatant, and hepatocytes preparations were used for subsequent experiments.

Periportal and pericentral subpopulations of wt hepatocytes were isolated by perfusion with digitonin and collagenase, as previously reported [START_REF] Braeuning | Differential gene expression in periportal and perivenous mouse hepatocytes[END_REF].

Cell culture

For primary culture of hepatocytes, livers were perfused three days after tamoxifen injection to Apc lox/lox mice, the cells were dispersed in William's medium (supplemented with 10% fetal bovine serum, penicillin-streptomycin, fungizone, 25nM dexamethasone, insulin 4 μg/mL and 1% BSA), and plated in wells coated with rattail collagen I (GIBCO).

Transfection assays

The cells were transfected 4h after plating, as described in the supporting materials.

xCELLigence assay

Hepatocyte adhesion and cell cycle progression were measured with the xCELLigence system (Roche). Cells were transfected with 100 nM LNA or mimic 4 h after plating, as described above (see supporting Materials). The impedance was then measured for 48 h.

Western-Blotting

The membrane with 50μg proteins was probed overnight with a primary antibody against the protein of interest (see Supporting Materials), and then incubated with a HRP-conjugated secondary antibody (Cell Signaling) for detection with the ECL system (Biorad).

In situ hybridization

Sections were deparaffinized and treated with 0.1 mg/ml proteinase K, then hybridized for one hour at 55°C with 100 nmol digoxigenin-labeled-LNA-scramble or LNA-34a (Exiqon). Signals were detected with anti-digoxigenin (Roche) and NBT/BCIP (Roche).

Samples processing for miRNA-seq:

Total RNA was extracted from purified hepatocytes six days after tamoxifen injection with Trizol® reagent (Life Technologies). For each set of conditions, we analyzed four distinct hepatocyte preparations by deep sequencing on an Illumina platform (Fasteris, Switzerland). The reads generated by the HiSeq were analyzed with miRDeep2 and miRAnalyzer software (See Supporting Materials).

Chromatin sonication and immunoprecipitation

The ChIP protocol was adapted from that of Nelson [START_REF] Nelson | Protocol for the fast chromatin immunoprecipitation (ChIP) method[END_REF], as previously described [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF] (See Supporting Materials).

Human samples

We included 44 patients treated for liver cancer at Cochin hospital in this study (Table S3). All tumor samples were frozen after surgery.

RNA extraction and RT-qPCR

Total RNA was extracted from purified hepatocytes with Trizol® reagent (Life Technologies). Levels of miRNA were determined on 2 ng total mRNA, with a specific Taqman miRNA assay (Applied Biosystems). Level of cyclin D1 and HNF-4α was determined on 100 ng total mRNA relative to 18S RNA (see Supporting Materials).

Statistical analysis

We assessed the significance of differences between two groups of samples in Wilcoxon tests if n<30 and Student tests if n>30. ANOVA was used to compare three groups of samples. P<0.05 was considered statistically significant.

RESULTS

MiR-34a is induced following β-catenin activation

We searched for candidate microRNAs regulated by β-catenin in hepatocytes, by deep-sequencing the miRNAs expressed in the pre-tumoral Apc deletion (Apc KO ) model, in which there is pan-lobular β-catenin overactivation, and comparing the results with those for wild-type (wt) hepatocytes. A statistical analysis showed that 123 miRNAs discriminated between Apc KO and wt hepatocytes (Tables S1 andS2), and that these miRNAs mostly targeted the Wnt/β-catenin pathway (Figure S1). We confirmed that miR-375 levels were significantly decreased by β-catenin activation (Table S2), as reported in HCCs with β-catenin mutation [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF]. We also identified 54 miRNAs generated from the imprinted Dlk1/Gtl2 locus as positive targets of β-catenin (Table S1). We discarded this locus for further studies, as this is not a relevant target of β-catenin in human HCCs (see the supplemental materials, Tables S1,S3 and Figure S2). We thus focused on miR-34a, which was induced by a factor of about three after β-catenin activation, with respect to wild-type hepatocytes, in the miRNAseq analysis (Table S1), and in qPCR (Figure 1A). This miRNA displayed the most significant induction following β-catenin activation after the miRNAs from the Dlk1/Gtl2 locus, and its overexpression in HCC with β-catenin mutation has already been reported [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF]. We collected 24 human tumors with a mutation of CTNNB1 leading to β-catenin activation, and 20 tumors without CTNNB1 mutation. This cohort covered the major etiologies observed for HCC (HBV, HCV, alcohol abuse) (Table S3). In our cohort of patients, miR-34a levels were higher in tumors than in normal tissue (by a factor of 2 in the absence of CTNNB1 mutation, and 5 in presence of CTNNB1 mutation, Figure 1B); this induction was significantly stronger in tumors with β-catenin mutations (p<0.005). The overexpression of miR-34a in tumors with mutations of β-catenin was correlated with a context of HCV infection or no particular risk factors (Figure 1C). The sample size was too small to obtain a significant difference in miR-34a overexpression as a function of alcohol abuse status (Figure 1C). The overexpression of miR-34a was confirmed by in situ hybridization in another panel of tumors with β-catenin mutation (Figure 1D). Thus, miR-34a is overexpressed in human HCCs with CTNNB1 mutations, and could constitute a major mediator of the oncogenic signal initiated by β-catenin activation in hepatocytes.

β-catenin directly regulates miR-34a expression

For confirmation of miR-34a regulation by β-catenin, we transfected isolated wt and Apc KO hepatocytes with a siRNA against β-catenin. β-catenin silencing decreased miR-34a levels by about 47% for wt hepatocytes and 33% for Apc KO hepatocytes (Figure 2A). We thus investigated whether β-catenin could directly control miR-34a expression, by ChIP experiments with an antibody against TCF-4 (Tcell factor 4), the main LEF/TCF factor associated with β-catenin in liver [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF], and an antibody against β-catenin. We measured the binding of TCF-4 and β-catenin to sites upstream from miR-34a identified as a TCF-4 binding site in our ChIP-seq data [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. βcatenin activation led to a significant increase in the binding of TCF-4 (Figure 2B) and β-catenin (Figure 2C) to a site 21kb upstream from miR-34a. β-catenin binding was also significantly increased following Apc depletion on a more proximal TCF-4 site (Figure S3). Overall, our data suggest that an excess of β-catenin could significantly increase miR-34a transcription in hepatocytes.

MiR-34a zonal redistribution after β-catenin overactivation

We then assessed the hepatic distribution of miR-34a by in situ hybridization.

Exceptions, such as cyclin D1 [START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF], are known, but the direct targets of β-catenin are generally expressed in pericentral (PC) hepatocytes, in which Wnt/β-catenin signaling is activated [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF]. Counterintuitively, in normal livers, miR-34a was found to be preferentially expressed in hepatocytes around the portal vein and, more particularly, in bile duct cells (Figure 3A). For confirmation of this prevalence of miR-34a in the periportal (PP) subpopulation, we isolated PP hepatocytes and PC hepatocytes separately, by injecting digitonin into the central vein and the portal vein, respectively. This differential perfusion also showed that PP samples were enriched in miR-34a (Figure 3B). The results were non-significant, since using this technique, biliary cells are mainly lost during washing steps. In addition, β-catenin overactivation led to a loss of miR-34a zonation, and to miR-34a expression of in all hepatocytes (Figure 3A). In conclusion, miR-34a appears to be expressed in hepatocytes throughout the liver only when β-catenin is overactivated.

We then searched the DIANA database (http://diana.imis.athenainnovation.gr/DianaTools/index.php?r=microT_CDS/index), a database with experimentally validated targets for miRNAs, for potential targets of miR-34a.

Number of potent targets have been identified (Table S4), but we focused on three interesting proteins: LEF-1, a transcription factor associated with β-catenin, caspase 2, recently identified as a direct target of miR-34a in the liver [START_REF] Meng | Epigenetic regulation of miR-34a expression in alcoholic liver injury[END_REF], and HNF-4α, which we recently showed to be an antagonist of β-catenin in the liver [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. These opposing activities result in a reciprocal physiological distribution in the liver, with miR-34a found in biliary cells (Figure 3A) and HNF-4α in hepatocytes, consistent with the hypothesis that miR-34a could repress HNF-4α expression in biliary cells [START_REF] Nagy | Expression of hepatic transcription factors during liver development and oval cell differentiation[END_REF].

MiR-34a inhibition improves HNF-4α transactivation capacity in hepatocytes displaying β-catenin-activation

We confirmed by western blotting that the levels of HNF-4α, caspase 2, and LEF-1 proteins (Figure 3C) were inversely correlated with miR-34a levels in Apc KO mice.

Similarly, the overexpression of miR-34a in human HCCs with CTNNB1 mutations was also associated with weaker immunostaining for HNF-4α in these tumors (Figure S4). In a previous study, we showed that β-catenin impaired the transcriptional activity of HNF-4α on a luciferase reporter gene under the control of an HNF-4αresponse element (FOP) [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. Having shown that β-catenin induces miR-34a, which could in turn regulate HNF-4α levels, we investigated the involvement of miR-34a in the antagonism between HNF-4α and β-catenin in Apc KO hepatocytes. An inhibitor of miR-34a based on locked nucleic acid technology (LNA-34a), which consists of the complementary sequence for miR-34a with a methylene group in the ribose, significantly activated the FOP reporter (Figure 3D). A mimic of miR-34a had no effect, probably due to the maximal miR-34a overexpression in Apc KO hepatocytes (Figure 3C). In conclusion, miR-34a represses HNF-4α transcriptional activity, and appears to be involved in the β-catenin/HNF-4α antagonism in our model of β-catenin overactivation.

MiR-34a is induced in tumors displaying β-catenin activation

We then assessed the level of miR-34a in liver tumours mutated for β-catenin in mice. We compared 35 tumors with seven normal tissues obtained from wt mice that had received tamoxifen injections. MiR-34a levels in these tumors were twice those in normal tissues (Figure 4A). HNF-4α mRNA levels in these tumors were also half those in normal tissues (Figure 4B), confirming its tumor suppressor role in the liver [START_REF] Lazarevich | Deregulation of hepatocyte nuclear factor 4 (HNF4)as a marker of epithelial tumors progression[END_REF][START_REF] Ning | Hepatocyte nuclear factor 4 alpha suppresses the development of hepatocellular carcinoma[END_REF]. Interestingly, the tumors with the highest miR-34a levels also had the lowest HNF-4α levels (Figures 4C and4D). The deregulation of miR-34a in these tumors was independent of p53 mRNA levels (Figure S5), despite miR-34a being a target of p53 [START_REF] He | A microRNA component of the p53 tumour suppressor network[END_REF]. Finally, we confirmed that overexpression of miR-34a in tumors with βcatenin overactivation, by in situ hybridization, was associated to a loss of HNF-4α (Figure 4E).

MiR-34a inhibition by a locked nucleic acid

These data highlight the unique oncogenic role of miR-34a in a context of βcatenin overactivation. This led us to try to block miR-34a activity with a LNA, which has proved promising in human liver disease [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF]. We assessed the efficacy of this approach in cultures of hepatocytes isolated from wt and Apc KO mice. No change in the morphology of hepatocytes transfected with LNAs was observed, on microscopy, 72 hours after transfection (data not shown). The efficacy of LNA-34a was assessed by cotransfection with a luciferase reporter construct with a miR-34a binding site in its 3'UTR. As expected, following cotransfection with LNA-34a, miR-34a activity was impaired, resulting in an increase in luciferase activity (about three times higher than that obtained after cotransfection with LNA-scramble, Figure 5A). The LNA efficacy was also confirmed by western blotting for HNF-4α: HNF-4α levels were higher in the presence of LNA-34a (Figure 5B).

LNA-34a impairs hepatocyte proliferation after β-catenin overactivation

We then investigated the effect of LNA-34a on the proliferation of hepatocytes. We used the xCELLigence system, which measures cell impedance in real-time, through the use of gold microelectrodes placed under each well. WT hepatocytes underwent one round of cell division, due to the proproliferative effect of tamoxifen and collagenase perfusion (Figure S6A). WT hepatocytes had a lower cell proliferation index than Apc KO hepatocytes (Figure S6A), confirming that an absence of Apc leads to hepatocyte hyperproliferation [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. Transfection with LNA-34a significantly impaired Apc KO hepatocyte proliferation (35% inhibition), but had no effect on wt hepatocytes (Figure 5C). Similar results were obtained with the Cell Titer assay, measuring the reduction of tetrazolium in formazan, a process dependent on NADPH (data not shown). The activity of lactate deshydrogenase in cell supernatant was not altered following LNA-34a transfection, suggesting an absence of LNA-34a toxicity (Figure S6B). This suggests that LNA-34a has a specific antiproliferative effect on hepatocytes displaying β-catenin overactivation. In addition, the LNA-34a transfected three days successively increased the amount of caspase-2 propeptide, as well as caspase-2L, its proapototic isoform, and also decreased the level of full-length PARP-1, suggesting its increased cleavage (Figure 5D). This was not observed for wt hepatocytes (Figure S6C). In vivo, the LNA-34a injected to mice only increased caspase-2L level in Apc KO mice (Figure S6D). In conclusion, LNA-34a promotes a pro-apoptotic signal in the Apc KO hepatocytes.

Cyclin D1 has been identified as a target of miR-34a [START_REF] Sun | Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest[END_REF]. We therefore assessed cyclin D1 levels after transfection with LNA-34a. Contrary to expectations, cyclin D1 protein and mRNA levels, at a lower extent, were decreased in the presence of LNA-34a (Figure 5E). However, this is consistent with the antiproliferative effects of LNA-34a on Apc KO hepatocytes (Figure 5C). The loss of cyclin D1 may be a consequence of the increase in HNF-4α amount in the presence of LNA-34a (Figure 5B), as HNF-4α has been shown to repress cyclin D1 [START_REF] Walesky | Hepatocyte-specific deletion of hepatocyte nuclear factor-4alpha in adult mice results in increased hepatocyte proliferation[END_REF] (Figure 6E). In agreement with this hypothesis, no change in cyclin D1 protein and mRNA levels was observed when Apc KO hepatocytes also lacking hnf-4α, were transfected with LNA-34a (Figure 6A).

LNA-34a had also a weaker antiproliferative effect on these hepatocytes isolated from a transgenic mouse model, in which both Apc and hnf4a had been deleted by Cre-Lox strategy (Figure 6B). The higher proliferation rate observed for hnf4/Apc KO hepatocytes in the presence of LNA-34a was associated to a minor induction of caspase-2L level, even if caspase-2 propeptide was efficiently increased (Figure 6C); PARP amount was also not altered in this case (Figure 6C). Finally, we realized ChIP experiments with an antibody against HNF-4α in Apc KO hepatocytes, and analyzed the binding of HNF-4α in the presence of LNA-34a on two binding sites identified in published ChIP-seq experiments done in HepG2 cells and in murine livers (Figure S7). We showed that the binding of HNF-4α to cyclin D1 promoter tended to increase following exposure to LNA-34a, in particular for peak 1 (Figure 6D). This finding corroborates that HNF-4α mediates the decrease in cyclin D1 levels in response to LNA-34a. In conclusion, following its overactivation, β-catenin binds to the miR-34a

promoter, thereby increasing its expression. MiR-34a in turn decreases the amount of HNF-4α, leading to increases in cyclin D1 transcription, and hepatocyte proliferation (Figure 6E).

MiR-34a inhibition reduces tumor progression in Apc KO mice

We thus developed a tumor suppressive strategy based on miR-34a inhibition, which could constitute the first targeted therapy for liver tumors displaying β-catenin activation. We assessed the impact of LNA-34a on liver tumor progression in an Apc KO context. Once the tumors became detectable on ultrasound scans, we injected LNA-34a (10 mg/kg) once weekly, and followed tumor development twice monthly, by 2D-ultrasound (Figure S8). We found that LNA-34a slowed the progression of tumors displaying β-catenin activation, to rates about half those for untreated tumors (Figure 7A andS8). This treatment also inhibited the development of new tumors, with only one new tumor on average occurring on LNA-34a treatment, versus four on LNAscramble treatment (Figure 7B). As expected, HNF-4α levels increased and cyclin D1 levels decreased in tumors treated with LNA-34a (Figure S9A). Even if cyclin D1 level was impaired, Ki-67 labeling was similar in tumors treated with LNA-34a, and in those treated with the scramble LNA (Figure S9B). Similar results were obtained with western-blots for phospho-Histone H3 and cyclin A labeling (data not shown).

However, LNA-34a promoted caspase 2 and 3 activation in tumors, in both westernblot and immunohistochemistry experiments, confirming its pro-apoptotic effect (Figures 7C and7E), with minor effects on non-tumoral liver (Figure 7D). Thus, treatment based on the use of LNA-34a could reduce tumor development through an increase in HNF-4α levels, leading to a loss of cyclin D1, and through a pro-apoptotic program associated with the induction of caspase 2 and caspase 3 cleavage.

DISCUSSION

The microRNA miR-34a has been widely described as a tumor suppressor.

Indeed, its expression is frequently impaired in various cancers following an hypermethylation of its promoter [START_REF] Lodygin | Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer[END_REF], including breast and prostate cancers [START_REF] Liu | The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44[END_REF][START_REF] Yang | MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1[END_REF].

A preclinical trial is currently underway with a mimic of miR-34a encapsulated into liposomes in various murine models of solid tumors (miRNATherapeutics, http://www.mirnarx.com/pipeline/mirna-MRX34.html). However, the role of miR-34a in liver tumorigenesis remains unclear. One study suggested that miR-34a level decrease in HCC [START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF], and is associated to migration and invasion [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF], but another two studies reported the induction of miR-34a in HCC samples [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF][START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF], in HCV+ sera [START_REF] Cermelli | Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease[END_REF], and in HCC cells in response to alcohol [START_REF] Meng | Epigenetic regulation of miR-34a expression in alcoholic liver injury[END_REF]. Our findings suggest that miR-34a is an oncogenic mediator in tumors displaying β-catenin activation, both in mice and humans, confirming the data reported by Pineau et al. [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF]. Our results could reconcile the data of Lou and Li, which suggested that miR-34a was lost in two thirds of the HCCs studied [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF][START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF]. These samples corresponded mostly to HBV + tumors, a subgroup with a lower frequency of CTNNB1 mutations [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF]. The other third of patients in their cohort, with HCV infection, displayed an increase in miR-34a expression, and this group is likely to be enriched in CTNNB1 mutations, according to transcriptomic classifications [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF]. In our cohort, tumors without β-catenin mutations displayed heterogeneity in terms of miR-34a amount. A greater understanding of this variability would be very useful,

In our study, we used a LNA approach to block cell cycle progression (Figure 5C), to decrease cyclin D1 protein levels (Figure 5E), and to induce caspase-2 activation (Figure 5D), in isolated Apc KO hepatocytes. The decrease in cyclin D1 protein levels was actually due to HNF-4α, because the invalidation of hnf4a in the Apc KO model resulted in an absence of change in cyclin D1 levels (Figure 6A), and a smaller decrease in cell proliferation (Figure 6B), in the presence of LNA-34a. In addition, LNA-34a increases HNF-4α protein level (Figure 5B), but also its transcriptional activity on a luciferase construct under the control of a HNF-4 responsive element (Figure 3D), and on cyclin D1 promoter (Figure 6D). Thus, HNF-4α, a tumor suppressor in the liver [START_REF] Lazarevich | Deregulation of hepatocyte nuclear factor 4 (HNF4)as a marker of epithelial tumors progression[END_REF], appears to be a key mediator of the effects of miR-34a in the Apc KO model (Figure 6E). More importantly, miR-34a emerges as a crucial component of the oncogenic signal induced by β-catenin in hepatocytes.

In normal livers, miR-34a was particularly expressed in bile duct cells (Figure 3A and 3B). This specific pattern of expression may hinder the accumulation of HNF-4α protein in the biliary cells, which is required for bile cell lineage patterning [START_REF] Nagy | Expression of hepatic transcription factors during liver development and oval cell differentiation[END_REF],

probably independently of β-catenin, which is not activated in normal bile duct cells [START_REF] Hu | Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells[END_REF]. The pattern of miR-34a expression is not consistent with the usual model, in which β-catenin targets are located near the central vein. Cyclin D1 is one exception to this model; its pattern of expression is not restricted to the PC zone [START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF].

Interestingly, the region bound to TCF-4/β-catenin located in miR-34a promoter contains no classical Wnt-responsive element, suggesting that the control on miR-34a is complex (Figure 2B). This complexity is lost when β-catenin is overactivated, as in HCCs.

The key finding of this study was the decrease in liver tumor growth afforded by the delivery of LNA-34a (Figures 7A andS8). Only a few studies to date have demonstrated the potency of miRNA-based approaches to cancer treatment.

However, the great advantage of this approach for HCC is that these molecules are preferentially delivered to the liver [START_REF] Roberts | Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice[END_REF]. The first authorization for clinical trials with miRNA-based therapies, concerns the LNA against miR-122 (miravirsen) for HCV treatment [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF]. The use of these modulators poses a number of challenges, particularly for their specificity. Our therapeutic approach, involving the inhibition of miR-34a, displays no toxicity (Figure S6B), and has the great advantage of restoring the tumor suppressor HNF-4α, targeting cyclin D1 and the metabolic pathways regulated by HNF-4α (Figure S9). In addition, this approach may also alter the antiinflammatory loop orchestrated by HNF-4α, controlling interleukin-6 production by hepatocytes [START_REF] Gougelet | MicroRNA-feedback loop as a key modulator of liver tumorigenesis and inflammation[END_REF][START_REF] Hatziapostolou | An HNF4alpha-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis[END_REF]. More importantly, the LNA-34a promotes a pro-apoptotic signal orchestrated by caspase 2 and 3 (Figure 7C-E). In conclusion, our data reveal that miR-34a inhibition mainly induces a pro-apototic program centered onto caspase-2, and could affect cell proliferation through cyclin D1 inhibition. However, the fact that Ki-67 and cyclin A labeling were not affected by LNA-34a treatment, suggests that miR-34a effects on cell proliferation are more complex.

In conclusion, despite the current preclinical trials being run by miRNAtherapeutic, our findings suggest that the precise differential diagnosis of HCC, including an assessment of the mutational status of β-catenin, for which miR-34a inhibitors would be more appropriate than miR-34a mimics, is vital. Using this approach, we hope to affect not only the tumor itself, but also its environment, through the first effective targeted therapy for one third of HCCs worldwide.

Figure 1 :

 1 Figure 1: miR-34a is induced following β-catenin activation in mouse

Figure 2 :

 2 Figure 2: miR-34a is induced following β-catenin activation, through the direct

Figure 3A -Figure 4 :

 3A4 Figure 3A-B: miR-34a induction following β-catenin activation is associated

Figure 5 :

 5 Figure 5: LNA-34a inhibits Apc KO hepatocyte proliferation, decreases cyclin D1

Figure 6 :Figure 7 :

 67 Figure 6: LNA-34a impact depends on HNF4α expression

ACKNOWLEDGMENTS

We thank Dr F. Gonzalez for the hnf4 -/-mice. We are grateful to Dr P. Bossard and M. Caüzac for LDH protocol and to Dr. A-F. Burnol for the gift of PARP-1 antibody.

SUMMARY BOX

What is already known on this subject ?

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and the third leading cause of death by cancer worldwide, due to the lack of effective treatments. We, and others, have shown that mutations of the CTNNB1 gene, encoding β-catenin, are observed in 15 to 40% of HCCs. HCCs with β-catenin mutation constitute one of the two subclasses of HCC, and are thought to follow a particular pattern of pathogenesis.

What are the new findings?

Using a transgenic mouse model developing tumors with β-catenin activation, we provide the first evidence of an oncogenic role of miR-34a in liver tumors with βcatenin mutations. An inhibitor against miR-34a slows tumor progression, and inhibits the development of new tumors in this model.

How might it impact on clinical practice in the foreseeable future?

These findings call into question the preclinical trial designed by miRNAtherapeutics, using miR-34a mimics to combat liver tumor growth. Instead, we provide here proof-of-concept that a locked nucleic acid-based inhibitor of miR-34a may be useful for treatment, exclusively in HCCs with β-catenin mutation.

STUDY APPROVAL

All procedures were carried out according to the French guidelines for the care and use of experimental animals. All animal studies were approved by the regional