miRNA biomarkers to predict risk of primary non-function of fatty allografts and drug induced acute liver failures

miRNA biomarkers to predict risk of primary non-function of fatty allografts and drug induced acute liver failures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article miRNA biomarkers to predict risk of primary non-function of fatty allografts and drug induced acute liver failures Juliette Schönberg, Jürgen Borlak This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4616493/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Primary non-function (PNF) of an allograft defines an irreversible graft failure and although rare, constitutes a life-threatening condition that requires high-urgency re-transplantation. Equally, drug induced acute liver failures (ALF) are seldom but the rapid loss of hepatic function may require orthotropic liver transplantation (OLT). Recently, we reported the development of a PNF-disease model of fatty allografts and showed that a dysfunctional Cori and Krebs cycle and inhibition of lactate transporters constitute a mechanism of PNF. We identified highly regulated miRNAs and their target genes and selected 15 miRNA-biomarker candidates for clinical validation. Our study aimed at their clinical validation. Additionally, we assessed their diagnostic value in ALF. We performed RT-qPCRs of 15 miRNA-biomarker candidates in well-documented PNF cases following OLT of fatty allografts. To assess specificity and selectivity, we compared their regulation in pre- and intraoperative liver biopsies and post-operative in blood samples of patients undergoing elective hepatobiliary surgery. Results: We confirmed regulation of 11 PNF-associated miRNAs in clinical PNF cases and found expression of miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p to correlate with the hepatic steatosis grade. Furthermore, we demonstrate selectivity and specificity for the biomarker candidates with opposite regulation of let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p in blood samples of patients following successful OLTs and/or liver resection. Strikingly, and based on 21 independent studies, eight PNF-associated miRNAs are also regulated in ALF. Conclusions: We report miRNAs highly regulated in PNF and ALF. Their common regulation in different diseases broadens the perspective as biomarker candidates for an identification of patients at risk for PNF and ALF. liver failure ischemia injury non-coding RNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Primary non-function (PNF) defines an irreversible graft failure without reasonable surgical or immunological causes [ 1 , 2 ]. It is a rare, but life-threatening condition and typically requires urgent re-transplantation (re-OLT). To better understand causes of PNF, we evaluated the reason, frequency and survival of re-OLT cases among 1,205 orthotopic liver-transplantation (OLT) which had been performed at our institution [ 3 ]. We considered a wide range of donor and recipient related clinical data. Overwhelmingly, fatty allografts were the main cause for PNF and were associated with excessive mortality after re-OLT. Although cold-ischemia and reperfusion injury potentially contributed to the risk of PNF, the Cox proportional-hazard regression analysis defined fatty liver allografts as the sole factor and was independently associated with worse outcome after re-OLT [ 3 ]. Importantly, non-alcoholic fatty liver disease (NAFLD) has reached pandemic proportions and some estimates suggest a prevalence of up to 90% in obese, 70% of overweight and about 25% in the general population [ 4 , 5 ]. Histologically, the presence of macrovesicular lipid droplets in > 5% of hepatocytes defines hepatic steatosis. Typically, pathologists distinguish between micro- and macrovesicular steatosis, and the occurrence of inflammation leading to non-alcoholic steatohepatitis (NASH) and different grades of fibrosis [ 6 , 7 ]. Although liver biopsies are the golden standard in the diagnosis of NAFLD [ 8 ], their use in a transplant setting is constrained for a number of reasons. First, biopsies harbor inherent risks of organ damage. Second, it is difficult to obtain specialized histological stains within the time frame of < 12h between organ procurement and OLT. Third, the procurement of donor organs and the subsequent transplantation frequently involves different hospitals, and the logistics of obtaining a pathology assessment in time can be demanding. Notwithstanding, liver biopsies are obtained from marginal organs with potentially suspicious macroscopic findings [ 9 ]. Given the significant shortage of high-quality donor allografts, the need arises to extend the criteria for inclusion of donors (ECD). The outcome of ECD liver transplantation has been the subject of independent reviews [ 10 , 11 ]. There is clear evidence for fatty liver allografts to be a major risk factor for PNF [ 3 , 12 , 13 ], and the question of how much fat is tolerable in allografts is the subject of a controversial debate. We recently reported an animal model to investigate mechanisms of fatty liver induced PNF [ 14 ] and found hepatic steatosis induced by a methionine choline deficient diet (MCD) to aggravate liver injury induced by the ischemia/reperfusion (IR) injury following OLT. Additionally, fatty allografts suffer from a dysfunctional TCA cycle with major implications for the metabolic competence of the liver. This includes a significant decline in energy/ATP production. Additionally, drug induced acute liver failure is defined by the rapid loss of hepatic functions without prior evidence of chronic liver disease. Estimates based on the United Network for Organ Sharing (UNOS) liver transplant database suggested that about 15% of liver transplants are due to drug hepatotoxicity of which acetaminophen overdose is a common cause [ 15 , 16 ]. Note ALF can be self-limiting but may progress with complete loss of hepatic function. Consequently, predicting patients which likely develop a life-threatening situation, and therefore require OLT, is an important unmet medical need. To identify biomarkers predictive of outcome, we performed genome wide scans in a PNF-disease model of fatty allografts[ 14 ]. The significantly regulated miRNAs have an established role in the regulation of hepatic lipid metabolism, injury and liver regeneration as well as programmed cell death and are mechanistically linked to PNF. In general, miRNAs are small noncoding RNAs (21-23nt) and confer translational repression [ 17 , 18 ]. Their role as transcriptional regulators in liver diseases is a hot topic [ 19 – 21 ], and miRNAs are of critical importance in the regulation of hepatic lipid synthesis and metabolism [ 22 ]. Correspondingly, their contribution to the pathophysiology of fatty liver disease is the subject of intense research [ 23 , 24 ], and miRNA biomarkers carry the potential to assist in an evaluation of the quality of donor allografts [ 25 ]. Based on findings from the PNF animal disease model, we questioned the clinical relevance of regulated miRNAs and given its unpredictable nature, evaluated PNF-associated miRNAs in formalin-fixed and paraffin-embedded archived tissue (FFPE) of clinical cases and compared the results to histologically normal liver resection material of patients undergoing elective surgery. Furthermore, to demonstrate selectivity, we compared their regulation in blood and liver tissue samples before, intra- and post-surgery for up to 3 days. Overall, we report a translational study, aimed at defining the clinical relevance of miRNA biomarker candidates derived from an animal model of PNF, and additionally questioned their diagnostic utility in ALF. Material and Methods Sample collection The basic patient characteristics are given in supplementary Table S1 . PNF and control liver resection material We obtained formalin-fixed and paraffin-embedded tissue (FFPE) blocks from the Pathology Institute of Hannover Medical School and assessed liver section material from 29 individual PNF cases and 11 controls. The controls are FFPE tissue blocks of histologically confirmed non-tumor (R0) resection material of patients diagnosed with liver metastasis. The PNF cases were mostly analyzed in duplicate samples and consisted of N = 22 fatty liver allografts and N = 7 non-fatty liver cases as determined by histopathology. The blocks were sectioned to 5 µm thickness and 10 sections of each block were combined for RNA analysis. The mean age of PNF and CLM patients was 48 and 65 years (supplementary Table S1 ) and 69% and 91%, respectively were males. Because of the gender disproportionate distributions, we investigated sex dependent regulation of miRNAs and as shown in supplementary Figure S1 , none are sex-related. Liver biopsies from transplant patients and elective surgeries We obtained biopsies from N = 7 donor-livers prior to OLT to enable an assessment of miRNA regulation following IR injury and its associated oxidative stress. Additionally, we obtained intraoperative (R0) resection biopsies from N = 10 patients following hilus occlusion of non-tumor material from patients diagnosed with CLM (N = 5) and cholangiocarcinoma (CCC, N = 5). The shock frozen biopsies enabled an assessment of cold- (organ storage, OLT) and warm-ischemia (Hilus occlusion during liver resection) and served as a surrogate endpoint to investigate the effects of organ storage and ischemia injury on the regulation of PNF associated miRNAs. All tissue samples were shock frozen and stored at -80°C to await further miRNA analysis. Plasma samples Matched blood samples of OLT patients (N = 7) were collected in EDTA tubes about 1h before surgery (t = -1h) and on day 1, 2 and 3 post-surgery. Additionally, blood samples of N = 7 patients undergoing elective liver surgery were obtained. As detailed above this group of patients served as an additional control to mimic reperfusion injury after opening of the intra-operative hilus occlusion. The sampling schedule was the same as for liver transplant patients, and the blood samples were centrifuged for 10 min at 2000 rpm, and the resultant plasma was stored at -80°C. None of the blood samples were hemolytic. RNA isolation FFPE tissue blocks On a rotary microtome, we prepared a total of 10 sections of 5 µm thickness from single tissue blocks and transferred the material into safe lock tubes. We extracted miRNA with the miRNeasy FFPE Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) and the sections were treated with 320µl deparaffinization solution (Qiagen) and incubated at 56°C for 3 min. Next, we added 240µl of PKD buffer, i.e., a buffer required for proteinase K digestion, vortexed the sample followed by centrifugation at 11,000g for 1 min. Thereafter, we pipetted 10 µl of proteinase K reagent into the lower (clear) phase of the solution and incubated the sample at 56°C on a Biometra TS1 Thermo Shaker (Analytik Jena AG, Jena, Germany) for 15 min followed by a second incubation step at 80°C for another 15 minutes. We transferred the lower, colorless phase into a safe lock tube and stored the sample on ice for 3 minutes and subsequently centrifuged at 20,000g in a Thermo Scientific Multifuge X1R (Thermo Fisher Scientific, Massachusetts, USA) for 15 minutes. Once again, we transferred the supernatant into a safe lock tube and added the DNase booster buffer equivalent to 1/10th of the volume in addition to 10µl DNase I stock solution. The samples were incubated at room temperature for 15 min followed by an addition of 500µl RBC buffer and vortexed to mix the lysate. Thereafter, we added 1750µl of EtOH (100%), vortexed the sample and transferred 700µl portions to the Rneasy MinElute spin columns. This was followed by the sequential elution with the RPE buffer according to the manufacturer’s recommendations. The eluates are discarded and the Rneasy MinElute spin columns are dried by centrifugation at 14,000 rpm. Finally, the spin columns are conditioned with 20µl Rnase free water and centrifuged at 14,000 rpm for 1 minute. We determined the RNA concentration by measuring the absorbance at 260nm with the Beckman coulter DU 730 Life Science UV/VIS Spectrophotometer (Beckman Coulter, California, USA) . The RNA concentrations ranged from 28.16ng/µl to 1801.85ng/µl. We calculated the ratio of 260 nm / 280 nm and 260nm / 230 nm absorption to obtain information about the purity of the RNA. Occasionally we performed RNA gel electrophoresis to examine the quality of the isolated RNA and the ribosomal bands. Plasma RNA isolation We isolated total RNA with the miRNeasy serum/plasma kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Briefly, we added 200µl plasma to 1000µl Qiazol and vortexed the sample for 60 seconds. The samples were incubated at room temperature for another 5 minutes. Next, we added 3.5 µl miRNeasy serum/plasma spike-in control working solution (= 5.6 x 10 8 copies) and 200µl chloroform (AppliChem, Darmstadt, Germany) and kept the sample at room temperature for 3 minutes. Subsequently, we centrifuged the samples at 12,000 g for 15 minutes and pipetted the upper phase, which contains RNA, into a new tube. We determined the volume (typically 700 µl) and added 100% EtOH at 1.5-fold excess of the initial volume and vortexed the sample. Then, 700µl portions were applied onto the Rneasy MinElute spin columns followed by an elution step with 700 µl RWT buffer, 500µl RPE buffer and 500µl of 80% EtOH. Finally, we centrifuged the spin columns at full speed (~ 12,000 rpm) for 5 minutes and eluted RNA with 14 µl Rnase free water. We added a spike in control, i.e. Cel-miR-39-3p ( Qiagen) to plasma samples to control the efficiency of the miRNA extraction. The amounts of RNA in plasma are very low, and therefore we could not measure RNA concentrations spectrophotometrically. Instead, we used 1.5 µl of the original RNA extract (see above) for reverse transcription. We prepared a standard curve by blotting different concentrations or copy numbers of the spike in control and the associated CT-values generated by real-time PCR. We calculated a linear regression and we obtained the following equation of the calibration curve: Y = -3.391*X + 48.3 Based on the constructed calibration curve the recovery and therefore efficiency of the extraction could be determined. The data are given as % recovery. RNA isolation from resection material We isolated total RNA from liver resection material of patients undergoing elective hepatobiliary surgery and biopsies taken from donor liver transplants (supplementary Table S1 , demographics) with the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The resection material was immediately shock frozen. Typically, we used 20-25mg frozen tissue from each patient and transferred the material into a vial for further processing. We added 700µl Qiazol, and the tissue was disintegrated with an Ultra-turrax t10 basic disperser tool (IKA, Staufen im Breisgau, Germany). Subsequently, we added 140µl chloroform (AppliChem, Darmstadt, Germany) and kept the sample at room temperature for 3 minutes. Next, the samples were centrifuged at 12,000 g for 15 minutes and we pipetted the upper phase, which contains the RNA, into a new tube. We determined the volume (typically 350 µl) and added 100% EtOH at 1.5-fold excess of the initial volume. We vortexed the sample and applied 700µl portions onto the Rneasy MinElute spin columns followed by elution steps with 350 µl RWT buffer, 500µl RPE buffer and 500µl of RPE according to the manufacturer’s recommendations. We centrifuge the spin columns at full speed (~ 12,000 rpm) for 2 minutes and eluted RNA with 30 µl Rnase free water. We determined the RNA concentrations spectrophotometrically by measuring the absorbance at 260nm with the Beckman coulter DU 730 Life Science UV/VIS Spectrophotometer (Beckman Coulter GmbH, Germany). We calculated the ratio of 260 nm / 280 nm and 260nm / 230 nm to obtain information about the RNA purity. The RNA concentration ranged from 608 ng/µl to 2689 ng/µl and we performed agarose gel electrophoresis to visualize ribosomal bands and to assess the quality of the isolated RNA. cDNA synthesis We initiated reverse transcription with the miScript II RT Kit (Qiagen, Hilden, Germany) . We prepared a master mix consisting of 5x miScript HiSpec buffer, 10x miScript Nucleics mix, miScript Reverse Transcriptase mix, Rnase-free water and template RNA. Typically, we used 1 µg of total RNA to initiate the reaction. In the case of blood/plasma samples the concentration of RNA is very low and typically could not quantify its concentration reliable. Therefore, we used 1.5µl of the eluate from the spin column for the isolation of RNA (see above) for RT. We performed the RT at 37°C for 60min followed by a cycle at 95°C for 5 min in the C1000 Touch Thermal cycler (Biorad, California, USA). Quantitative PCR of 15 PNF associated miRNAs Tissue RNA extracts: We performed qPCR with the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) . The reaction mix consisted of 12.5µl 2x QuantiTect SYBR Green PCR Master Mix, 2.5µl 10x miScript SYBR Universal Primer, 6.5µl Rnase-free water, 2.5µl 10x miScript Primer Assay and 1µl tissue derived template cDNA (~ 3ng). The total reaction volume is 25µl. Blood samples: We added 200µl of water to the entire cDNA prepared from individual plasma samples (approximately 20 µl) and used 4µl of cDNA template and 3.5µl water to the PCR-Mix to initiate the reaction as detailed above. We performed the PCR on a C1000 Touch Thermal cycler and a CFX96 Real-Time system (Biorad, California, USA) with settings described below (see Table 1 ). We used the Bio-Rad CFX Manager 3.1 software (Biorad) to analyse the data and to visualize the amplification curves. Given in supplementary Table S2 and S3 are the conditions of the PCR reactions and the primer sequences. Table 1 PNF regulated miRNAs in ALF, severe drug induced liver injury and fatty liver disease. PNF regulated miRNAs of the present study Independent confirmation of PNF regulated miRNAs in various pathological conditions Serum / Tissue PubMed miRNA 122-5p miRNA 122-3p acute liver failure and spontaneous remission from ALF S↑, T↑ [ 51 ] severe drug induced liver injury (DILI) progressing to ALF S↑ [ 57 ] Steatosis, NASH S↑, T↓ [ 62 , 74 ] miRNA 125b-5p HBV-ACLF T↓ [ 52 , 75 ] ALF, Regulator of cell death T↓ [ 76 ] sDILI / ALF S↑ [ 57 ] NASH S↑ [ 62 ] miRNA 192-5p sDILI / ALF S↑ [ 57 ] NAFLD, NASH S↑ [ 62 , 74 ] Apoptosis T↓ [ 77 ] miRNA 27b-3p sDILI / ALF S↑ [ 57 ] Steatosis S↑ [ 74 ] miRNA 103a-3p sDILI / ALF S↑ [ 57 ] Steatosis T↑ [ 78 ] miRNA 194-5p sDILI / ALF S↑ [ 57 ] We compared PNF associated miRNAs to published findings for acute liver failure and severe drug induced liver injury (DILI) cases. Note the DILI cases are mainly due to acetaminophen overdose. S = Serum, T = Tissue, P = Plasma ↑=upregulation, ↓=downregulation. Data-analysis We applied the 2 −(∆∆CT) method to calculate changes of disease regulated miRNAs using the following formula ∆CT = CT (miRNA of interest) – CT (reference gene) ∆∆CT = ∆CT (patient sample) – ∆CT (healthy control) Fold change = 2^ -(∆∆CT) We used RNU6B as a reference gene to determine the ∆Ct value of a miRNA of interest of either fresh or FFPE liver tissue material. In the case of plasma extracts the mean of miRNA16-5p and Cel-miR-39-3p served as a reference gene. To evaluate the regulation of miRNAs among PNF cases and to determine fold changes, we calculated the ∆CT values of N = 11 individual controls and used the average ∆CT for comparisons with individual PNF cases. Additionally, we investigated the regulation of miRNAs in the circulation pre- and post-surgery (up to 3 days) and compared the results with tissue extracts of the same patients. We applied the Shapiro–Wilk method to test for normality. Depending on the data distribution, we used the paired/unpaired t-test or the non-parametric Mann-Whitney-Test or Wilcoxon matched-pairs signed rank test. A * denotes a significant p -value < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. We performed all statistical computations with the GraphPad Prism software version 8.4.3. Search for miRNA gene targets Based on a fatty allograft PNF disease model [ 14 ] we performed genome wide scan to identify target genes of significantly regulated miRNAs in PNF. Among the highly regulated miRNAs, we focused on those with an established role in hepatic lipid metabolism, liver injury and regeneration as well as programmed cell death. Based on genome wide miRNA scans, we selected 15 miRNAs that were highly regulated and performed gene ontology annotations to convert rat miRNAs into their human orthologues using the g-profiler program [ 26 ]. Subsequently, we queried the miRNet public repository to identify potential genes targeted by the selected miRNAs [ 27 ]. We compared the list of potential targets with significantly regulated genes which we identified in fatty allograft failing livers of the rat (PNF study) and searched for common targets. This defined 2,307 genes, and we visualized the miRNA-target gene networks with the Cytoscape software (U.S. National Institute of General Medical Sciences, NIGMS). Additionally, we evaluated the biological functions of the regulated target genes with the gene ontology tool Metascape [ 28 ] and David database [ 29 ] and created visual networks. Rat Serum Details regarding the animal study are given in our recent publication (see Kulik et al., 2024 [ 14 ]), and the study is reported in accordance with ARRIVE guidelines. Ethical approval was granted by the animal welfare ethics committee of the State of Lower Saxony, Germany (“Lower Saxony State office for Consumer Production and Food Safety” [LAVES]). The approval ID is Az: 33.14-42502-04-13/1258. All methods were carried out in accordance with relevant guidelines and regulations. Blood samples were obtained from (CTx) and fatty allograft recipients (MTx) post OLT on day 1, 3, 7 and 14. We prepared serum from whole blood using standard procedures and performed a genome wide search for regulated miRNA [ 14 ]. This defined differentially expressed miRNAs in the circulation of fatty allograft recipient animals, and we selected 15 miRNAs for their time dependent regulation and clinical validation. Results Rationale for miRNA selection Based on findings from a disease model of fatty allograft associated PNF [ 14 ], we selected 15 highly regulated miRNAs with known functions in the control of lipid metabolism, apoptosis, acute liver failure and liver regeneration (Table 1 ). Shown in Fig. 1 are H&E-stained liver sections of PNF allografts with varying degree of hepatic steatosis, inflammation and necrosis. Case A1 is a 43 year (y) old male who received a fatty allograft. Histology of the first allograft evidenced marked centrilobular and subcapsular map-like necrosis excessive inflammatory infiltrates, shrunken and vacuolated hepatocytes and macrovesicular steatosis (Case A1). Case A2 is a 58y old female. Note the marked macrovesicular steatosis, the centrilobular necrosis, the partial destruction of portal fields and extra-hepatic bile duct necrosis. Panel B1 and B2 refers to a 39y old female. Here, histology revealed subtotal necrosis indicative for excessive reperfusion injury. A further example relates to a 40y old female, and the liver section in C1 shows mixed micro- and macrovesicular steatosis, parenchymal necrosis and ischemia/reperfusion injury. Panel C2 refers to a 57y old male patient, and this liver section shows primarily macrovesicular steatosis, perivenular cholestasis with occasional lymphocytic infiltrates. Depicted in Fig. 1 panel D1 is the case of a 60y old male with marked portal inflammatory infiltrates, extensive lobular necrosis and macrovesicular steatosis of the fatty allograft. Case D2 refers to a 49y old female, and the allograft shows excessive macrovesicular steatosis, fresh hemorrhage, centrilobular necrosis and acute fatty liver dystrophy. PNF-associated miRNAs To validate PNF associated miRNAs, we analyzed fifty-nine FFPE tissue blocks of 29 PNF cases and compared the data to 11 individual controls, i.e. morphological normal liver resection material obtained in the course of an elective hepatobiliary surgery (supplementary Table S1 ). Furthermore, we addressed the question whether the degree of hepatic steatosis influenced their regulation. First, we considered the expression of the house keeping gene RNU 6B. Its expression did not differ between controls and PNF cases irrespective of the degree of hepatic steatosis (Fig. 2 A). Therefore, the selection of the housekeeping gene is justified and could be used as a “normalizer” in qPCR assays. Second, we considered the interpatient variability in the expression of individual miRNAs and found all miRNAs to behave similar (supplementary Table S4). Therefore, we exclude sampling bias as a possible confounder. Third, we computed the 2 −(∆∆CT) -values of miRNAs among PNF cases and found 11 out of 15 miRNAs to be significantly regulated (Fig. 2 B&C, non-regulated miRNAs in supplementary Figure S2 A). Except for miRNA-125a-5p and miRNA-195-5p (Fig. 2 B) the miRNAs were repressed in expression when compared to morphologically normal tissue as exemplified for miRNA-26a-5p and miRNA-27b-3p which were repressed to about 30% of controls (p < 0.0001, Fig. 2 C). Note, the latter two miRNAs were highly repressed in 80% and 85% of cases. Fourth, we addressed the question whether the degree of hepatic steatosis influenced expression of PNF-associated miRNAs. While for the majority of PNF regulated miRNAs the expression remained alike (supplementary Figure S2 B), we found miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p to be significantly influenced by the degree of hepatic steatosis (Fig. 2 D). The results suggest that hepatic steatosis aggravated the repression of these miRNAs. Fifth, we compared the regulation of PNF associated miRNAs in clinical samples to findings obtained from the animal study and the results were comparable (Table 2 ). For instance, in rat liver and human PNF cases miRNA-122-5p was repressed to 35% and 34% of controls. Notwithstanding, there are also significant differences in PNF associated miRNA regulations between human cases and the animal model. Specifically, let-7b-5p, miRNA-125a-5p, miRNA-126-3p, miRNA-194-5p and miRNA-195-5p were oppositely regulated between clinical cases and the animal model and the changes were more pronounced in the animal PNF-model. Table 2 Regulation of PNF associated miRNAs in human and rat liver tissue. PNF rat Mean, 95%-CI PNF human Mean, 95%-CI PNF human p-value Let-7b-5p 15% (13–18%) 211% (100–302%) 0.19 miRNA-19b-3p 11% (10–12%) 51% (41–70%) 0.16 miRNA-23b-3p 4% (4–5%) 65% (48–89%) 0.02 miRNA-26a-5p 6% (6–7%) 30% (24–36%) < 0.0001 miRNA-27b-3p 48% (25–100%) 30% (21–36%) < 0.0001 miRNA-103a-3p 6% (6–7%) 5% (43–59%) 0.002 miRNA-122-3p 4% (4–4%) 28% (17–52%) 0.0004 miRNA-122-5p 35% (29–45%) 34% (30–73%) 0.006 miRNA-125a-5p 4% (4–4%) 166% (130–215%) 0.04 miRNA-125b-5p 3% (3–3%) 53% (42–66%) 0.009 miRNA-126-3p 3% (3–3%) 154% (119–195%) 0.27 miRNA-192-5p 21% (18; 25) 55% (35–67%) 0.006 miRNA-194-5p 7% (6; 8) 105% (77–137%) 0.59 miRNA-195-5p 4% (4; 4) 228% (172–301%) 0.02 miRNA-455-3p 8% (7; 8) 46% (33–54%) 0.02 The data are mean and 95%-CI-values. Ischemia injury To assess the effects of ischemia-injury on the regulation of PNF associated miRNAs, we evaluated their expression in biopsy of liver allografts (N = 7, supplementary Table S5A) prior to transplantation. Additionally, we obtained intraoperative biopsies after hilus occlusions during hepatic surgery (N = 10, supplementary Table S5B) and compared the regulation of miRNAs of donor liver biopsies during organ storage to intra-operative liver biopsies taken from patients undergoing elective hepatobiliary surgery. The data shown in Fig. 3 are ∆CT values (Fig. 3 ) for significantly upregulated miRNAs following hilus occlusion. For instance, miRNA-122-5p was nearly 3-fold upregulated in liver tissue following ischemia injury (median: 2.6-fold, Fig. 3 B). This miRNA is a well-known marker of liver injury. Independent research demonstrated miRNA-122-5p to be highly enriched in the nucleus of liver cells and to block activity of the cell survival oncomiR miR-21 at the posttranscriptional level [ 30 ]. Since ischemia injury is associated with marked cellular damage, its release into circulation is expected. Indeed, we found blood borne miRNA-122-5p to be 19-fold upregulated on day 1 post-surgery and this represents a > 600% increase of this miRNA when compared to its induced tissue expression (see Fig. 4 ). Additionally, tissue expression of let-7b-5p, miRNA-125b-5p and miRNA-194-5p were significantly upregulated by about 3-fold, and these miRNAs are known to augment inflammation and hepatic stellate cell activation. For comparison, the data of non-significantly regulated miRNAs are given in supplementary Figure S3. miRNA in the systemic circulation following reperfusion injury of clinical cases Ischemia/reperfusion (IR) injury is an unavoidable process in hepatic surgery and to determine whether the selected miRNA biomarkers are also regulated in response to IR injury, we investigated their regulation in blood samples taken prior to (T = 0) and post-surgery on day 1, 2 and 3. We evaluated 14 patients of which one-half were OLT cases, and the other half consisted of elective surgeries for primary or secondary liver malignancies (supplementary Table S6). We obtained serial blood samples from the same patients and determined CT-values by the 2 −(∆∆−CT) -method. We used the average CT-values of miRNA-16-5p and Cel-miRNA-39-3p as reference genes (Fig. 4 A), and independent research demonstrated the advantageous of applying the average of two reference genes for data analysis [ 31 – 33 ]. The time course of individual blood borne miRNAs are shown in Fig. 4 B-G, and 6 out of 15 PNF associated miRNAs, i.e., 27b-3p, 122-3p,122-5p,125b-5p,192-5p and miRNA-194-5p were significantly upregulated in plasma by a range of 7- 26-fold post-surgery. Importantly, we observed opposite regulation of tissue (Fig. 2 ) and blood borne miRNAs and this highlights their sensitivity to ischemia-reperfusion injury (IRI). All regulated miRNAs returned to pre-surgery or even below T = 0 expression values on day 3 post-surgery and unchanged miRNAs are given in supplementary Figure S4. However, miRNA-126-3p remained consistently repressed (Fig. 4 H) and experimental research demonstrated this miRNA to be a target Hoxb6. This transcription factor controls expression of SOX9 in liver progenitor cells which are destined to replace damage cells following CCL4 liver injury of mice [ 34 ]. Therefore, a regulatory loop exists between miRNA-126-3p, Hoxb6 and SOX9, and it is tempting to speculate that repressed miRNA-126-3p serum levels in clinical samples signify delayed liver regeneration. Additionally, we searched for blood borne miRNAs either regulated in OLT or tumor associated surgery. Depicted in supplementary Figure S5, panel A are miRNAs which are explicitly regulated in tumor liver resection cases, i.e. miRNA-19b-3p, miRNA-125a-5p and miRNA-126-3p and these function in wound repair and fibrosis, liver regeneration and metabolic disease. For instance, overexpression of miRNA-125a-5p supports liver regeneration [ 35 ]. Conversely, miRNA-27b-3p and miRNA-194-5p are specifically regulated in OLT (supplementary Figure S5, panel B) and these function in inflammation and rejection of the graft [ 36 ]. To evaluate different grades of hepatic steatosis on the regulation of blood borne miRNA, we compared plasma samples of patients diagnosed with mild to moderate steatosis (N = 7) to cases of marked steatosis (N = 7). Obviously, the number of patients is small, but even so, miRNA-103a-3p reached statistical significance and this miRNA promotes hepatic steatosis by repressing the expression of palmitoyl-CoA oxidase [ 37 ]. Furthermore, the expression of miRNA-26a-5p, miRNA-27b-3p, miRNA-103a-3p and miRNA-122-5p tended to be higher in cases of marked steatosis (supplementary Figure S6), however, did not reach statistical significance. Serum miRNAs in a rat fatty allograft OLT model We recently reported the development of a PNF fatty allograft disease model [ 38 ]and determined the regulation of 15 miRNAs in blood samples of rats following liver transplantation on days 1, 3, 7 and 14 post-surgery (Fig. 5 ). We compared their expression in serum of non-transplanted Chow-fed controls to serum values following OLT of healthy allografts. The data are shown as fold changes and we observed upregulation of miRNA-27b-3p, miRNA-122-3p, miRNA-122-5p, miRNA-125a-5p, miRNA-126-3p, miRNA-192-5p, miRNA-194-5p and miRNA-195-5p (Fig. 5 A, range 25 to 2-fold). Conversely, miRNA-7b-5p, miRNA-19b-3p, miRNA-23b-3p, miRNA-26a-5p, miRNA-125b-5p and miRNA-455-3p were downregulated (Fig. 5 B, range 22 to 1.5-fold). Although the latter miRNAs were below the expression of Chow-fed controls, it is obvious that their expression increased with time, thus implying improved liver function following OLT. Additionally, we compared the regulation of PNF associated serum miRNAs in donor animals on a CHOW and MCD diet for 7 and 14 days (Fig. 6 , left panels). Essentially, we observed mild increases of these miRNAs (range 2 to 4-fold) in MCD fed animals. This demonstrates their fatty liver associated regulation. Subsequently, we compared the regulation of PNF associated miRNAs in serum of rats following OLT of healthy and fatty allografts. Shown in the right panels of Fig. 6 are serum miRNAs regulated in recipient animals following OLT of fatty allografts. The data are fold changes by comparing it to healthy allografts, and we divided the results into up-regulated (Fig. 6 A) and down regulated (Fig. 6 B) miRNAs post OLT for up to 7 days. We observed marked induction of miRNA-122-3p and miRNA-122-5p, i.e. 48- and 10-fold induced, respectively in fatty allografts on day 7 post-OLT. Similarly, we observed a 19-fold increased expression of miRNA-194-5p in fatty allografts on day 7 post OLT. Furthermore, miRNA-126-3p, miRNA-192-5p and miRNA-194-5p were upregulated on day 1, 3 and 7 but downregulated on day 14 post-OLT. Conversely, Let-7b-5p, miRNA-27b-3p, miRNA-103-3p, miRNA-125a-5p, miRNA-125b-5p were down regulated in recipient animals of fatty allografts. Meanwhile, miRNA-19b-3p, miRNA-23b-3p and miRNA-26a-5p were unchanged up to 7 days post-OLT but declined thereafter (Fig. 6 B). Except for miRNA-122, all serum miRNAs were significantly down regulated on day 14 post-OLT (Fig. 6 ). Thus, fatty allograft OLTs are hallmarked by repression of PNF associated miRNAs and the results are similar to clinical samples (Fig. 2 ) on day 14 post-OLT. miRNA gene-target networks in PNF We performed a genome wide scan to identify genes regulated in PNF cases [ 14 ], and for this purpose compared the transcriptomes of rat liver following OLT of healthy donor allografts (CTx) to PNF cases. This revealed 2450 differential expressed genes (DEGs) of which 2215 or nearly 91% were repressed. Therefore, PNF caused an unprecedented repression of the transcriptome and involved various components of the general transcription machinery including the CAAT enhancer binding proteins, TATA-Box binding protein associated factors, i.e. TAF-proteins, and various liver enriched transcription factors (HNF4alpha and FOXA3, Fig. 7 A) [ 39 , 40 ]. Strikingly, the BRD4 bromodomain and extra-terminal domain transcriptional activator is highly induced (> 10-fold) and was recently shown to be key player in the global loss of activity of the transcriptional machinery in damaged livers [ 41 ]. Typically, BRD4 binds to acetylated lysine residues of the chromatin (super-enhancers) and supports transcriptional activation of genes. Furthermore, we queried the miRnet public data base to search for experimentally proven targets of 15 PNF associated miRNAs and compared the results to DEGs identified in PNF livers. We focused on target genes coding for hepatic lipid metabolism, liver injury and regeneration as well as programmed cell death and report results for 363 target genes. Depicted in Fig. 7 are the network for autophagy (panel 7B), apoptotic signaling (panel 7C), response to endoplasmic reticulum stress (panel 7D), response to hypoxia (panel 7E), mitochondrial organization (panel 7F) and response to cytokine (panel 7G). Note, the majority of target genes are repressed, and 4/5 of the regulated genes are experimentally proven targets while the remaining are predicted targets. Discussion Based on findings from a preclinical liver transplant model [ 14 ], we aimed at validating fatty allografts associated miRNAs predictive for PNF. We confirmed clinical significance for 11 miRNAs, of which 9 and 2, respectively were down and upregulated (Table 2 and Fig. 2 ). Our study revealed the significant relationship between the degree of hepatic steatosis and the repression of miRNA-27b-3p, miRNA-122-3p, miRNA-125b-5p and miRNA-192-5p in liver tissue of clinical PNF-cases (Fig. 2 D). Note under hypoxic conditions, and through upregulation of the transcription factor PPARγ, miRNA-27b plays an essential role in lipid metabolism [ 42 , 43 ] while silencing of miRNA-125b-5p promotes liver fibrosis in nonalcoholic fatty liver disease via integrin α8-mediated activation of the RhoA signaling pathway [ 44 ]. Furthermore, repressed miRNA-192-5p aggravates lipid deposition by controlling the expression of stearoyl-CoA desaturase 1 [ 45 , 46 ]. Additionally, we explored the regulation of PNF associated miRNAs in liver biopsies taken prior to OLT and intraoperatively following hilus occlusion. We found 4 miRNAs (let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p) significantly upregulated when biopsies following hilus occlusion were compared to T0 liver biopsies (Fig. 3 ). Moreover, we identified 6 up- and 1 downregulated miRNA in post-surgery blood samples of successfully performed OLTs and tumor liver resection cases (Fig. 4 ). Importantly, these miRNAs were oppositely regulated when compared to PNF cases (Fig. 2 ). Therefore, we demonstrate selectivity and specificity and clinical relevance for the majority of the miRNAs. Table 3 summarizes the 15 miRNAs and their regulation in FFPE-PNF-tissue (Fig. 2 ), pre- and intraoperative liver biopsies (Fig. 3 ) and blood samples taken from patients which underwent elective hepatobiliary surgery (Fig. 4 ). Table 3 Regulation of miRNAs in human fatty allograft associated PNF cases, in T0 liver biopsies of healthy allografts prior to OLT and hepatectomies of tumor resection. miRNA Tissue expression in fatty allograft associated PNF OLT healthy allografts and hepatectomy of neoplasms Plasma Liver tissue miRNA 122-5p ↓ ** ↑ *** ↑ * miRNA 125b-5p ↓ ** ↑ ** ↑ *** miRNA 27b-3p ↓ **** ↑ ** ns miRNA 122-3p ↓ *** ↑ *** ns miRNA 192-5p ↓ ** ↑ *** ns miRNA 26a-5p ↓ **** ns ns miRNA 23b-3p ↓ * ns ns miRNA 103a-3p ↓ ** ns ns miRNA 455-3p ↓ * ns ns miRNA 125a-5p ↑ * ns ns miRNA 195-5p ↑ * ns ns miRNA 194-5p ns ↑ ** ↑ * miRNA 126-3p ns ↓ * ns Let 7b-3p ns ns ↑ ** miRNA 19b-3p ns ns ns In Table 1 , we summarize the various functions of PNF associated miRNAs in the control of lipid metabolism, acute liver failure, IR-injury and liver regeneration. The regulation of miRNA-122 and its two mature products, i.e. miRNA-122-3p and − 5p is an interesting example [ 47 ]. Although abundantly expressed in the liver, miRNA-122-3p is not significantly regulated in liver tissue resection material (Fig. 3 ); however, is highly upregulated in blood samples following surgery (Fig. 4 ). On the first day post-surgery, its regulation ranged between 0.7 and 270-fold across individual patients and this miRNA serves as a marker of liver cell damage. In contrast, miRNA-122-5p is mildly but significantly upregulated in intra-operative biopsy samples following hilus occlusion (median = 2.6-fold) and markedly increased in blood samples of the same patients (median = 20-fold). miRNA-122 is essential for liver metabolic homeostasis and lipid metabolism. It exerts anti-inflammatory and anti-fibrotic properties and blocks viral replication in hepatocytes [ 48 , 49 ]. Notwithstanding one report suggests liver injury-induced release of miRNA-122 to stimulate pulmonary inflammation [ 50 ]. Typically, its expression is low in serum but highly upregulated during liver injury. Interestingly, in patients with spontaneous recovery from acute liver failure miRNA-122 is significantly upregulated in serum and liver tissue when compared to no recovered patients. This implies an important role of this miRNA in instructing liver regeneration [ 51 ]. A further example relates to miRNA-125b-5p which was reported to alleviate acute liver failure by regulating the Keap1/Nrf2/HO-1 pathway [ 52 ]. Furthermore, this miRNA protects from reperfusion injury by inhibiting TRAF6 and NF-κB signaling [ 53 ]. Unlike preclinical PNF cases, let-7b-5p is regulated in human liver tissue, but not in blood samples, and this miRNA inhibits cell proliferation [ 54 ]. Notwithstanding, one study identified repressed let-7b blood levels in children diagnosed with progressive familial intrahepatic cholestasis [ 55 ]. In vitro, this miRNA inhibits hepatic stellate cell activation and therefore plays a role in fibrosis [ 56 ]. A recent review summarized significantly regulated miRNAs in human acute liver failure (ALF) cases [ 57 ]. The review is based on 21 independent studies and primarily describes findings for acetaminophen overdose and drug induced liver injury (DILI) cases as well as viral liver disease. Of the ALF serum and plasma regulated miRNAs, 53% are common to our study, i.e. 8/15 miRNA, and this demonstrates relevance of these miRNAs in acute liver failure across independent clinical studies. Although the causes of ALF and PNF are different in nature, i.e. drug induced versus fatty allograft associated PNFs, the results underscore the clinical relevance of the selected miRNAs and their utility as commonly regulated biomarkers in PNF and ALF. Table 1 compiles miRNAs commonly regulated between clinical ALF and fatty allograft associated PNF cases and highlights their basic function in liver biology. For instance, miRNA-27b-3p regulates mitochondrial biogenesis [ 58 ] and targets several key lipid-metabolism genes [ 59 ]. This miRNA is highly repressed in fatty allograft associated PNF cases (Fig. 2 ) and given its role in mitochondrial biogenesis, its repression might be regarded as an adaptive response. Indeed, an inverse relationship exists between miRNA-27b expression and mitochondria content [ 58 ]. Similarly, de novo lipogenesis can be inhibited by miRNA-27a. This miRNA alleviates obesity-initiated NAFLD by repressing the expression of fatty acid synthase and stearoyl-CoA desaturase [ 60 ]. Furthermore, miRNA-27b-5p inhibits PPARγ driven lipogenesis [ 61 ]. A further example relates to an identification of circulating miRNAs in NAFLD patients. Specifically, Pirola and co-workers investigated serum microRNAs among liver biopsy proven NAFLD cases and healthy controls [ 62 ]. Of the 84 investigated miRNAs, blood borne miRNA-122, miRNA-192, miRNA-19a and miRNA-19b, miRNA-125b proved to be of diagnostic value. In the present study miRNA-122-5p, miRNA-192-5p, miRNA-125b-5p were highly significantly repressed among fatty allograft associated PNF cases and their regulation was influenced by the hepatic lipid content (Fig. 2 D). Unlike liver biopsy and serum findings for NAFLD patients [ 62 ] miRNA-19b-3p was not significantly regulated in fatty allograft associated PNF cases (supplementary Figure S2 , panel A). The role of miRNA-192-5p in human diseases is the subject of a recent review and there is evidence for this miRNA to effect energy metabolism [ 46 ]. Downregulation of miRNA 192 causes hepatic steatosis through upregulation of sterol regulatory element binding transcription factor 1 [ 63 ]. In the present study, miRNA-192-5p was markedly repressed among fatty allograft associated PNF cases. Its regulation was influenced by the hepatic lipid content (Fig. 2D5) and correlated with the degree of steatosis. Conversely, miRNA-192-5p is significantly upregulated in blood samples of patients following OLT of healthy allografts and patients undergoing hepatectomy (Fig. 4 ). Moreover, the importance of the HNF4α-miRNA-194/192 signaling axis in maintaining hepatic cell function was demonstrated in liver-specific Hnf4a-null (Hnf4aΔH) mice [ 64 ] and miRNA-192-5p and miRNA-194-5p are localized in a cluster. Note both miRNAs were significantly upregulated in plasma samples following hepatic surgery (Fig. 4 ) and this demonstrates its diagnostic relevance for distinguishing PNF from liver regeneration cases. Another miRNA linked to liver regeneration is miRNA-26a. This miRNA is significantly repressed in PNF cases but abundantly expressed in OLT biopsy and blood samples of patients undergoing elective liver surgery (Figs. 3 and 4 ). Independent research demonstrated that the growth factor termed augmenter of liver regeneration (ALR) induces expression of miRNA 26a and stimulated cell proliferation via the microRNA-26a/Akt/cyclin D1 signaling pathway [ 65 ]. Conversely, miRNA-26 influences the cross-talk between mdm2 and p53 and its repression stimulates mdm2 expression which inhibits p53 activity [ 66 ]. Another study demonstrated down-regulation of microRNA-26a to promote mouse hepatocyte proliferation during liver regeneration [ 67 ]. Therefore, repressed miRNA-26 supports liver regeneration and can be regarded as an adaptive response to impair programmed cell death. Liver regeneration is supported by the upregulation of miRNA-125a-5p, and its overexpression in the human liver cell line HL-7702 increased cell viability significantly [ 68 ]. In the present study, miRNA-125a-5p was one of the two significantly increased miRNAs (Fig. 2 ), and we consider its upregulation in fatty allograft associated PNF cases as an attempt to stimulate liver regeneration. Notwithstanding, miRNA-195-5p was also significantly upregulated and this miRNA promotes hepatic stellate cell activation and liver fibrosis by suppressing PTEN expression in a mouse model of liver damage [ 69 ]. Furthermore, down-regulation of miR-23b stimulated TGF-β1/Smad3 signaling during the termination stage of liver regeneration [ 70 ] and therefore contributes to impaired liver regeneration. Consistent with its function miR-23b is repressed in fatty allograft associated PNF cases (Fig. 2 ). Lastly, we observed repressed plasma miRNA-126-3p in post-surgery blood samples of OLTs and tumor liver resection cases (Fig. 4 ). This miRNA suppresses inflammation in endothelial cells [ 71 ], is significantly repressed in higher grade NAFLD patients [ 72 ], and its repression impairs liver regeneration in mice following partial hepatectomy [ 73 ]. Based on their specific regulation by the grade of hepatic steatosis, we propose miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p as a panel of diagnostic miRNAs to predict fatty allograft associated PNF. Their validation in prospective clinical trials is warranted. In addition, miRNA-26a-5p is highly regulated in most PNF cases (80%) and therefore is a biomarker candidate worthwhile for in depth validation. Conclusions We report an identification of miRNAs significantly associated with fatty allograft associated PNFs. Our findings warrant clinical validation to demonstrate their prognostic value. Study limitations We would like to highlight the following study limitations. First, PNF is a rare and unpredictable event, and therefore it is difficult to design a prospective study. Thus, our findings are based on archived tissue materials. Second, given the retrospective nature of the study, we are unable to control bias, i.e. the outcome was known prior to study initiation. However, we do not consider this bias to be of critical importance for an identification of PNF biomarkers. Third, we were unable to obtain sufficient number of intraoperative liver biopsies following OLT of healthy allograft. Fourth, although we included all fatty allograft associated PNF cases among 1,200 OLTs performed at our institution, we report a single center study. Nonetheless, the power analysis showed the number of cases to be sufficient to determine statistical significance. Future studies should be based on randomized clinical trials. Abbreviations ALF acute liver failure ATP adenosine triphosphate CCC cholangiocellular carcinoma cDNA complementary DNA CLM colorectal liver metastasis CT crossing threshold DEGs differential expressed genes DILI drug induced liver injury DNA desoxyribonuclein acid EAD early allograft dysfunction ECD extended criteria donors EDTA ethylene diamine tetraacetic acid ER endoplasmatic reticulum EtOH ethanol FFPE formalin-fixed and paraffin-embedded tissue H&E hematoxylin and eosin staining HCC hepatocellular carcinoma IRI ischemia-reperfusion injury CTx normal liver recipient rats MCD methionine/choline deficient diet miRNA micro ribonucleic acid MTx fatty liver recipient animals NAFLD non-alcoholic fatty liver disease NASH non-alcoholic steatohepatitis OLT orthotopic liver transplantation PNF primary non-function qPCR quantitative polymerase chain reaction RNA ribonucleic acid Rpm rounds per minute RT reverse transcription TCA tricarboxylic acid Y years Declarations Ethics approval and consent to participate We obtained approval from the Ethics Committee of the Hannover Medical School (8582_BO_S_2019, 8368_Bo_K_2019 and 7506_Bo_K_2017) for the use of archived PNF tissue blocks, fresh liver resection material and blood samples from patients undergoing elective hepatobiliary surgery. Informed consent was obtained from all subjects and/or their legal guardian(s). All methods were performed in accordance with the relevant guidelines and regulations. Details regarding the animal study are given in our recent publication (see Kulik et al., 2024 [14] ), and the study is reported in accordance with ARRIVE guidelines. Ethical approval was granted by the animal welfare ethics committee of the State of Lower Saxony, Germany (“Lower Saxony State office for Consumer Production and Food Safety” [LAVES]). The approval ID is Az: 33.14-42502-04-13/1258. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its supplementary files. Competing interests The authors declare that they have no competing interests. Funding Lower Saxony Ministry of Culture and Sciences and the Volkswagen Foundation, Germany to JB (25A.5-7251-99-3/00). Authors' contributions JS collected the samples and performed miRNA analysis and qPCR measurements. JB performed the histopathology study. Both authors analyzed the data. JS prepared the figures and supported the writing of the manuscript. JB wrote the final manuscript. Both authors reviewed and approved the manuscript. Acknowledgements We thank Dr. Ulf Kulik and the Clinic for General, Visceral and Transplant Surgery of Hannover Medical School for providing biopsies and blood samples and the Institute of Pathology for supplying FFPE tissue blocks of PNF cases. We gratefully acknowledge the technical support of Gabi Onken in the histopathology work. References Lock JF, Schwabauer E, Martus P, Videv N, Pratschke J, Malinowski M, Neuhaus P, Stockmann M (2010) Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. 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J Appl Toxicol 40:1683–1693 Soronen J, Yki-Jarvinen H, Zhou Y, Sadevirta S, Sarin A, Leivonen M, Sevastianova K, Perttila J, Laurila P, Sigruener A, Schmitz G, Olkkonen VM (2016) Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease. Physiol Rep 4:e12661. 10.14814/phy2.12661 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresS1S6.docx SupplementaryTablesS1S6.docx Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 11 Sep, 2024 Reviews received at journal 31 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers invited by journal 02 Aug, 2024 Editor assigned by journal 23 Jul, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 21 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4616493","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317373745,"identity":"fe6414cf-dc20-410b-92ce-b4999247dfff","order_by":0,"name":"Juliette Schönberg","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Juliette","middleName":"","lastName":"Schönberg","suffix":""},{"id":317373746,"identity":"1051152b-1f55-4167-bbc9-052c46aabea3","order_by":1,"name":"Jürgen Borlak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABKElEQVRIie3RsUrDQBjA8e84uC6XukYifYaDQEsw1AdxsQSyZfANCsJNia7mLTrW7UIgWQ5dDzJUKYhDh4hLxQrmqqLoiY4O94cELuHHfckB2Gz/MAaABAjAb+uwe0I/XrtmAp9J/HfyXvk7GfWya7GW0GN1try7n18djsApHo7n4eTME0i130mQ1qxIFWAm62GQyya5mPYjL5fxJD89wsG5YTAVg6BtR1RMfIc3yWyxYp7DS59JIB41k2KjyeKW+M/8MpkJ6j9pcqDJxkxKqgdTBC8RF5oM9S4DRjti+PwgraDcky7elTFBGY+2ZN/h8cCV6CRITX+Mo5tVFUb9usLtIx9vB2scHtKdFBdqbdjmNTfqbuTrKaDpj6Br3F3YcAg2m81mA3gBDSRrABo5I5UAAAAASUVORK5CYII=","orcid":"","institution":"Hannover Medical School","correspondingAuthor":true,"prefix":"","firstName":"Jürgen","middleName":"","lastName":"Borlak","suffix":""}],"badges":[],"createdAt":"2024-06-21 09:42:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4616493/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4616493/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-024-05129-3","type":"published","date":"2024-10-18T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60615233,"identity":"4eefe8d7-43e0-49d9-985b-5cd2f263f33b","added_by":"auto","created_at":"2024-07-18 20:11:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10300773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathology of fatty allograft associated PNF cases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepicted are 7 individual H\u0026amp;E-stained liver sections of PNF cases (panel B1 and B2 stems from the same individual) with various grades of hepatic steatosis, inflammation, necrosis and fatty liver dystrophy. A description of the individual cases is given in the result section.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/18dad39b19c1741c2c62bbaf.png"},{"id":60615228,"identity":"594ce3fc-5582-44f9-b0e3-9d84fe3a27c5","added_by":"auto","created_at":"2024-07-18 20:11:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":804851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of miRNAs in liver tissue of fatty allograft associated PNF cases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs were extracted from formalin-fixed, paraffin-embedded tissue sections (FFPE). The data are 2\u003csup\u003e-(∆∆CT)\u003c/sup\u003e-values for significantly regulated miRNAs. We used Shapiro–Wilk normality test and unpaired t-test or Mann-Whitney-Test. Shown are median and p-values. \u003cstrong\u003e(A) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eexpression of the\u003cstrong\u003e \u003c/strong\u003eRNU6B housekeeping gene is similar in control and PNF cases. \u003cstrong\u003e(B) \u003c/strong\u003emiRNA-125a-5p and miRNA-195-5p are significantly upregulated in liver tissue of fatty allograft associated PNF cases. \u003cstrong\u003e(C) \u003c/strong\u003emiRNA-23b-3p, miRNA-26a-5p, miRNA-27b-3p, miRNA-103a-3p, miRNA-122-3p, miRNA-122-5p, miRNA-125b-5p, miRNA-192-5p and miRNA-455-3p are significantly repressed in PNF cases.\u003cstrong\u003e (D) \u003c/strong\u003eHepatic steatosis grade dependent\u003cstrong\u003e \u003c/strong\u003eregulation of miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p among PNF cases. Note, significance is calculated from ∆CT-values (p=0.03, p=0.02, p=0.04\u003cstrong\u003e, \u003c/strong\u003ep=0.01, p=0,02).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/cd92699234f876b3fbcb3983.png"},{"id":60615229,"identity":"186577be-0eb0-413d-9d25-1086a9a9cf60","added_by":"auto","created_at":"2024-07-18 20:11:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":543316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIschemia injury regulated miRNAs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs were extracted from T0 liver biopsies, i.e. prior to OLT and intraoperative following hilus occlusion in the course of hepatectomy. The data are ∆CT-values for significantly regulated miRNAs. Importantly, subtracting the high abundance\u003cstrong\u003e \u003c/strong\u003eRNU6B reference gene from low\u003cstrong\u003e \u003c/strong\u003eabundance\u003cstrong\u003e \u003c/strong\u003emiRNA transcripts yielded negative ∆CT values. We compared expression of a given miRNA in T0 biopsies (organ storage prior to OLT, = cold ischemia) to its expression following hilus occlusion (= warm ischemia) and this caused increased expression of let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p. Note, a reduction in the ∆CT value implies an increase in transcript expression. We used Shapiro–Wilk normality test and unpaired t-test (p-values: p=0.01, p=0.03, p=0.0007\u003cstrong\u003e, \u003c/strong\u003ep=0.02).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/592c4970dc19d68440a02672.png"},{"id":60616375,"identity":"4adbf992-7e53-4f58-9733-9550d13d10ab","added_by":"auto","created_at":"2024-07-18 20:19:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":910137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlood-borne miRNAs after OLT of healthy allografts and tumor liver resections.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs were extracted from plasma prior to and post-surgery. The data are 2\u003csup\u003e-(∆∆CT)\u003c/sup\u003e-values for significantly regulated miRNAs. Except for miRNA-126-3p all miRNAs are upregulated on day 1 post-surgery and decline thereafter. We used Shapiro–Wilk normality test and paired t-test or Wilcoxon matched-pairs signed rank test.\u003cstrong\u003e (A)\u003c/strong\u003e Constant expression of the housekeeping gene miRNA-16-5p and Cel-miRNA-39-3p in pre- and post-surgery. \u003cstrong\u003e(B-G)\u003c/strong\u003e Upregulated: miRNA-27b-3p, miRNA-122-3p, miRNA-122-5p, miRNA-125b-5p, miRNA-192-5p and miRNA-194-5p. \u003cstrong\u003e(H\u003c/strong\u003e) Downregulated miRNA-126-3p.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/334364c1215dd7419485a32a.png"},{"id":60615232,"identity":"520d6260-b593-48d1-81b5-15214640498f","added_by":"auto","created_at":"2024-07-18 20:11:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":362673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiRNAs in the systemic circulation after OLT of healthy allografts in a rat liver transplantation model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs were extracted from rat serum post-surgery on days 1, 3, 7 and 14. The data are fold changes relative to non-transplanted controls. Depicted in panel A and B, respectively are predominantly up- and down regulated miRNAs following liver transplantation of healthy allografts.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/7def4652784b4dfe52bbdec9.png"},{"id":60615234,"identity":"e0206f54-e792-43ed-bb83-ce3a3394a2a1","added_by":"auto","created_at":"2024-07-18 20:11:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1289132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of serum miRNAs after OLT of fatty allografts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMiRNAs were extracted from rat serum samples of donor animals on day 7 and 14 (left panel). Furthermore, we show their regulation in recipient animals, post-surgery on days 1, 3, 7 and 14. The data are fold changes by comparing their expression in serum samples of healthy and recipients of fatty allografts. Depicted in panel A and B, respectively are predominantly up- and down regulated miRNAs following liver transplantation of fatty allografts.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/ae54efb1c301c6fe7a857afa.png"},{"id":60615237,"identity":"63d66eef-9b7d-49eb-b2b8-49f2eeb93c1b","added_by":"auto","created_at":"2024-07-18 20:11:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5496245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of miRNA-gene regulatory networks in rat fatty liver associated PNF cases\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe performed genomics in healthy allografts and fatty allografts associated PNF cases. Shown are miRNA-gene networks in failing livers following fatty allograft transplantation. \u003cstrong\u003e(A) \u003c/strong\u003eRegulation of transcription factors and transcriptional repressors \u003cstrong\u003e(B)\u003c/strong\u003e Regulation of miRNA-gene networks. Shown are Cytoscape visualized networks of autophagy. \u003cstrong\u003e(C)\u003c/strong\u003e Apoptotic signaling pathway. \u003cstrong\u003e(D)\u003c/strong\u003e Response to endoplasmic reticulum stress. \u003cstrong\u003e(E)\u003c/strong\u003eResponse to hypoxia. \u003cstrong\u003e(F) \u003c/strong\u003eMitochondrial organization. \u003cstrong\u003e(G) \u003c/strong\u003eResponse to cytokine.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/d45010b480f5ee8d86462009.png"},{"id":67149128,"identity":"4e85892d-2983-4ca2-a65b-8dae2ddaaffd","added_by":"auto","created_at":"2024-10-21 16:12:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25228909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/ff7ec5fa-f068-4905-9588-ca49cbcffa88.pdf"},{"id":60615236,"identity":"d7718275-9bfe-43d9-a7d5-fc8e24569fed","added_by":"auto","created_at":"2024-07-18 20:11:14","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":4690694,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresS1S6.docx","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/cc70a672f8de36f520936079.docx"},{"id":60615235,"identity":"ffa58b21-4cd9-4f39-9c91-d9150d1ff465","added_by":"auto","created_at":"2024-07-18 20:11:14","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":31541,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesS1S6.docx","url":"https://assets-eu.researchsquare.com/files/rs-4616493/v1/270f63011e261fb9a661ef19.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"miRNA biomarkers to predict risk of primary non-function of fatty allografts and drug induced acute liver failures","fulltext":[{"header":"Background","content":"\u003cp\u003ePrimary non-function (PNF) defines an irreversible graft failure without reasonable surgical or immunological causes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is a rare, but life-threatening condition and typically requires urgent re-transplantation (re-OLT).\u003c/p\u003e \u003cp\u003eTo better understand causes of PNF, we evaluated the reason, frequency and survival of re-OLT cases among 1,205 orthotopic liver-transplantation (OLT) which had been performed at our institution [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. We considered a wide range of donor and recipient related clinical data. Overwhelmingly, fatty allografts were the main cause for PNF and were associated with excessive mortality after re-OLT. Although cold-ischemia and reperfusion injury potentially contributed to the risk of PNF, the Cox proportional-hazard regression analysis defined fatty liver allografts as the sole factor and was independently associated with worse outcome after re-OLT [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, non-alcoholic fatty liver disease (NAFLD) has reached pandemic proportions and some estimates suggest a prevalence of up to 90% in obese, 70% of overweight and about 25% in the general population [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Histologically, the presence of macrovesicular lipid droplets in \u0026gt;\u0026thinsp;5% of hepatocytes defines hepatic steatosis. Typically, pathologists distinguish between micro- and macrovesicular steatosis, and the occurrence of inflammation leading to non-alcoholic steatohepatitis (NASH) and different grades of fibrosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although liver biopsies are the golden standard in the diagnosis of NAFLD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], their use in a transplant setting is constrained for a number of reasons. First, biopsies harbor inherent risks of organ damage. Second, it is difficult to obtain specialized histological stains within the time frame of \u0026lt;\u0026thinsp;12h between organ procurement and OLT. Third, the procurement of donor organs and the subsequent transplantation frequently involves different hospitals, and the logistics of obtaining a pathology assessment in time can be demanding. Notwithstanding, liver biopsies are obtained from marginal organs with potentially suspicious macroscopic findings [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the significant shortage of high-quality donor allografts, the need arises to extend the criteria for inclusion of donors (ECD). The outcome of ECD liver transplantation has been the subject of independent reviews [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. There is clear evidence for fatty liver allografts to be a major risk factor for PNF [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and the question of how much fat is tolerable in allografts is the subject of a controversial debate.\u003c/p\u003e \u003cp\u003eWe recently reported an animal model to investigate mechanisms of fatty liver induced PNF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and found hepatic steatosis induced by a methionine choline deficient diet (MCD) to aggravate liver injury induced by the ischemia/reperfusion (IR) injury following OLT. Additionally, fatty allografts suffer from a dysfunctional TCA cycle with major implications for the metabolic competence of the liver. This includes a significant decline in energy/ATP production.\u003c/p\u003e \u003cp\u003eAdditionally, drug induced acute liver failure is defined by the rapid loss of hepatic functions without prior evidence of chronic liver disease. Estimates based on the United Network for Organ Sharing (UNOS) liver transplant database suggested that about 15% of liver transplants are due to drug hepatotoxicity of which acetaminophen overdose is a common cause [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Note ALF can be self-limiting but may progress with complete loss of hepatic function. Consequently, predicting patients which likely develop a life-threatening situation, and therefore require OLT, is an important unmet medical need.\u003c/p\u003e \u003cp\u003eTo identify biomarkers predictive of outcome, we performed genome wide scans in a PNF-disease model of fatty allografts[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The significantly regulated miRNAs have an established role in the regulation of hepatic lipid metabolism, injury and liver regeneration as well as programmed cell death and are mechanistically linked to PNF.\u003c/p\u003e \u003cp\u003eIn general, miRNAs are small noncoding RNAs (21-23nt) and confer translational repression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Their role as transcriptional regulators in liver diseases is a hot topic [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and miRNAs are of critical importance in the regulation of hepatic lipid synthesis and metabolism [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Correspondingly, their contribution to the pathophysiology of fatty liver disease is the subject of intense research [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and miRNA biomarkers carry the potential to assist in an evaluation of the quality of donor allografts [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on findings from the PNF animal disease model, we questioned the clinical relevance of regulated miRNAs and given its unpredictable nature, evaluated PNF-associated miRNAs in formalin-fixed and paraffin-embedded archived tissue (FFPE) of clinical cases and compared the results to histologically normal liver resection material of patients undergoing elective surgery. Furthermore, to demonstrate selectivity, we compared their regulation in blood and liver tissue samples before, intra- and post-surgery for up to 3 days.\u003c/p\u003e \u003cp\u003eOverall, we report a translational study, aimed at defining the clinical relevance of miRNA biomarker candidates derived from an animal model of PNF, and additionally questioned their diagnostic utility in ALF.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThe basic patient characteristics are given in supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePNF and control liver resection material\u003c/h2\u003e \u003cp\u003eWe obtained formalin-fixed and paraffin-embedded tissue (FFPE) blocks from the Pathology Institute of Hannover Medical School and assessed liver section material from 29 individual PNF cases and 11 controls. The controls are FFPE tissue blocks of histologically confirmed non-tumor (R0) resection material of patients diagnosed with liver metastasis. The PNF cases were mostly analyzed in duplicate samples and consisted of N\u0026thinsp;=\u0026thinsp;22 fatty liver allografts and N\u0026thinsp;=\u0026thinsp;7 non-fatty liver cases as determined by histopathology. The blocks were sectioned to 5 \u0026micro;m thickness and 10 sections of each block were combined for RNA analysis.\u003c/p\u003e \u003cp\u003eThe mean age of PNF and CLM patients was 48 and 65 years (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and 69% and 91%, respectively were males. Because of the gender disproportionate distributions, we investigated sex dependent regulation of miRNAs and as shown in supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, none are sex-related.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLiver biopsies from transplant patients and elective surgeries\u003c/h2\u003e \u003cp\u003eWe obtained biopsies from N\u0026thinsp;=\u0026thinsp;7 donor-livers prior to OLT to enable an assessment of miRNA regulation following IR injury and its associated oxidative stress. Additionally, we obtained intraoperative (R0) resection biopsies from N\u0026thinsp;=\u0026thinsp;10 patients following hilus occlusion of non-tumor material from patients diagnosed with CLM (N\u0026thinsp;=\u0026thinsp;5) and cholangiocarcinoma (CCC, N\u0026thinsp;=\u0026thinsp;5). The shock frozen biopsies enabled an assessment of cold- (organ storage, OLT) and warm-ischemia (Hilus occlusion during liver resection) and served as a surrogate endpoint to investigate the effects of organ storage and ischemia injury on the regulation of PNF associated miRNAs. All tissue samples were shock frozen and stored at -80\u0026deg;C to await further miRNA analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlasma samples\u003c/h2\u003e \u003cp\u003eMatched blood samples of OLT patients (N\u0026thinsp;=\u0026thinsp;7) were collected in EDTA tubes about 1h before surgery (t = -1h) and on day 1, 2 and 3 post-surgery. Additionally, blood samples of N\u0026thinsp;=\u0026thinsp;7 patients undergoing elective liver surgery were obtained. As detailed above this group of patients served as an additional control to mimic reperfusion injury after opening of the intra-operative hilus occlusion. The sampling schedule was the same as for liver transplant patients, and the blood samples were centrifuged for 10 min at 2000 rpm, and the resultant plasma was stored at -80\u0026deg;C. None of the blood samples were hemolytic.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eRNA isolation FFPE tissue blocks\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eOn a rotary microtome, we prepared a total of 10 sections of 5 \u0026micro;m thickness from single tissue blocks and transferred the material into safe lock tubes. We extracted miRNA with the miRNeasy FFPE Kit according to the manufacturer\u0026rsquo;s instructions \u003cem\u003e(Qiagen, Hilden, Germany)\u003c/em\u003e and the sections were treated with 320\u0026micro;l deparaffinization solution \u003cem\u003e(Qiagen)\u003c/em\u003e and incubated at 56\u0026deg;C for 3 min. Next, we added 240\u0026micro;l of PKD buffer, i.e., a buffer required for proteinase K digestion, vortexed the sample followed by centrifugation at 11,000g for 1 min. Thereafter, we pipetted 10 \u0026micro;l of proteinase K reagent into the lower (clear) phase of the solution and incubated the sample at 56\u0026deg;C on a Biometra TS1 Thermo Shaker \u003cem\u003e(Analytik Jena AG, Jena, Germany)\u003c/em\u003e for 15 min followed by a second incubation step at 80\u0026deg;C for another 15 minutes. We transferred the lower, colorless phase into a safe lock tube and stored the sample on ice for 3 minutes and subsequently centrifuged at 20,000g in a Thermo Scientific Multifuge X1R \u003cem\u003e(Thermo Fisher Scientific, Massachusetts, USA)\u003c/em\u003e for 15 minutes. Once again, we transferred the supernatant into a safe lock tube and added the DNase booster buffer equivalent to 1/10th of the volume in addition to 10\u0026micro;l DNase I stock solution. The samples were incubated at room temperature for 15 min followed by an addition of 500\u0026micro;l RBC buffer and vortexed to mix the lysate. Thereafter, we added 1750\u0026micro;l of EtOH (100%), vortexed the sample and transferred 700\u0026micro;l portions to the Rneasy MinElute spin columns. This was followed by the sequential elution with the RPE buffer according to the manufacturer\u0026rsquo;s recommendations. The eluates are discarded and the Rneasy MinElute spin columns are dried by centrifugation at 14,000 rpm. Finally, the spin columns are conditioned with 20\u0026micro;l Rnase free water and centrifuged at 14,000 rpm for 1 minute. We determined the RNA concentration by measuring the absorbance at 260nm with the Beckman coulter DU 730 Life Science UV/VIS Spectrophotometer \u003cem\u003e(Beckman Coulter, California, USA)\u003c/em\u003e. The RNA concentrations ranged from 28.16ng/\u0026micro;l to 1801.85ng/\u0026micro;l. We calculated the ratio of 260 nm / 280 nm and 260nm / 230 nm absorption to obtain information about the purity of the RNA. Occasionally we performed RNA gel electrophoresis to examine the quality of the isolated RNA and the ribosomal bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlasma RNA isolation\u003c/h2\u003e \u003cp\u003eWe isolated total RNA with the miRNeasy serum/plasma kit \u003cem\u003e(Qiagen, Hilden, Germany)\u003c/em\u003e according to the manufacturer\u0026rsquo;s recommendations. Briefly, we added 200\u0026micro;l plasma to 1000\u0026micro;l Qiazol and vortexed the sample for 60 seconds. The samples were incubated at room temperature for another 5 minutes. Next, we added 3.5 \u0026micro;l miRNeasy serum/plasma spike-in control working solution (=\u0026thinsp;5.6 x 10\u003csup\u003e8\u003c/sup\u003e copies) and 200\u0026micro;l chloroform \u003cem\u003e(AppliChem, Darmstadt, Germany)\u003c/em\u003e and kept the sample at room temperature for 3 minutes. Subsequently, we centrifuged the samples at 12,000 g for 15 minutes and pipetted the upper phase, which contains RNA, into a new tube. We determined the volume (typically 700 \u0026micro;l) and added 100% EtOH at 1.5-fold excess of the initial volume and vortexed the sample. Then, 700\u0026micro;l portions were applied onto the Rneasy MinElute spin columns followed by an elution step with 700 \u0026micro;l RWT buffer, 500\u0026micro;l RPE buffer and 500\u0026micro;l of 80% EtOH. Finally, we centrifuged the spin columns at full speed (~\u0026thinsp;12,000 rpm) for 5 minutes and eluted RNA with 14 \u0026micro;l Rnase free water.\u003c/p\u003e \u003cp\u003eWe added a spike in control, i.e. Cel-miR-39-3p (\u003cem\u003eQiagen)\u003c/em\u003e to plasma samples to control the efficiency of the miRNA extraction. The amounts of RNA in plasma are very low, and therefore we could not measure RNA concentrations spectrophotometrically. Instead, we used 1.5 \u0026micro;l of the original RNA extract (see above) for reverse transcription. We prepared a standard curve by blotting different concentrations or copy numbers of the spike in control and the associated CT-values generated by real-time PCR. We calculated a linear regression and we obtained the following equation of the calibration curve: Y = -3.391*X\u0026thinsp;+\u0026thinsp;48.3\u003c/p\u003e \u003cp\u003eBased on the constructed calibration curve the recovery and therefore efficiency of the extraction could be determined. The data are given as % recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation from resection material\u003c/h2\u003e \u003cp\u003eWe isolated total RNA from liver resection material of patients undergoing elective hepatobiliary surgery and biopsies taken from donor liver transplants (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, demographics) with the miRNeasy mini kit \u003cem\u003e(Qiagen, Hilden, Germany)\u003c/em\u003e according to the manufacturer\u0026rsquo;s recommendations. The resection material was immediately shock frozen. Typically, we used 20-25mg frozen tissue from each patient and transferred the material into a vial for further processing. We added 700\u0026micro;l Qiazol, and the tissue was disintegrated with an Ultra-turrax t10 basic disperser tool \u003cem\u003e(IKA, Staufen im Breisgau, Germany).\u003c/em\u003e Subsequently, we added 140\u0026micro;l chloroform \u003cem\u003e(AppliChem, Darmstadt, Germany)\u003c/em\u003e and kept the sample at room temperature for 3 minutes. Next, the samples were centrifuged at 12,000 g for 15 minutes and we pipetted the upper phase, which contains the RNA, into a new tube. We determined the volume (typically 350 \u0026micro;l) and added 100% EtOH at 1.5-fold excess of the initial volume. We vortexed the sample and applied 700\u0026micro;l portions onto the Rneasy MinElute spin columns followed by elution steps with 350 \u0026micro;l RWT buffer, 500\u0026micro;l RPE buffer and 500\u0026micro;l of RPE according to the manufacturer\u0026rsquo;s recommendations. We centrifuge the spin columns at full speed (~\u0026thinsp;12,000 rpm) for 2 minutes and eluted RNA with 30 \u0026micro;l Rnase free water. We determined the RNA concentrations spectrophotometrically by measuring the absorbance at 260nm with the Beckman coulter DU 730 Life Science UV/VIS Spectrophotometer (Beckman Coulter GmbH, Germany). We calculated the ratio of 260 nm / 280 nm and 260nm / 230 nm to obtain information about the RNA purity. The RNA concentration ranged from 608 ng/\u0026micro;l to 2689 ng/\u0026micro;l and we performed agarose gel electrophoresis to visualize ribosomal bands and to assess the quality of the isolated RNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ecDNA synthesis\u003c/h2\u003e \u003cp\u003eWe initiated reverse transcription with the miScript II RT Kit \u003cem\u003e(Qiagen, Hilden, Germany)\u003c/em\u003e. We prepared a master mix consisting of 5x miScript HiSpec buffer, 10x miScript Nucleics mix, miScript Reverse Transcriptase mix, Rnase-free water and template RNA. Typically, we used 1 \u0026micro;g of total RNA to initiate the reaction. In the case of blood/plasma samples the concentration of RNA is very low and typically could not quantify its concentration reliable. Therefore, we used 1.5\u0026micro;l of the eluate from the spin column for the isolation of RNA (see above) for RT. We performed the RT at 37\u0026deg;C for 60min followed by a cycle at 95\u0026deg;C for 5 min in the C1000 Touch Thermal cycler \u003cem\u003e(Biorad, California, USA).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR of 15 PNF associated miRNAs\u003c/h2\u003e \u003cp\u003eTissue RNA extracts: We performed qPCR with the miScript SYBR Green PCR Kit \u003cem\u003e(Qiagen, Hilden, Germany)\u003c/em\u003e. The reaction mix consisted of 12.5\u0026micro;l 2x QuantiTect SYBR Green PCR Master Mix, 2.5\u0026micro;l 10x miScript SYBR Universal Primer, 6.5\u0026micro;l Rnase-free water, 2.5\u0026micro;l 10x miScript Primer Assay and 1\u0026micro;l tissue derived template cDNA (~\u0026thinsp;3ng). The total reaction volume is 25\u0026micro;l.\u003c/p\u003e \u003cp\u003eBlood samples: We added 200\u0026micro;l of water to the entire cDNA prepared from individual plasma samples (approximately 20 \u0026micro;l) and used 4\u0026micro;l of cDNA template and 3.5\u0026micro;l water to the PCR-Mix to initiate the reaction as detailed above.\u003c/p\u003e \u003cp\u003eWe performed the PCR on a C1000 Touch Thermal cycler and a CFX96 Real-Time system \u003cem\u003e(Biorad, California, USA)\u003c/em\u003e with settings described below (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We used the Bio-Rad CFX Manager 3.1 software \u003cem\u003e(Biorad)\u003c/em\u003e to analyse the data and to visualize the amplification curves. Given in supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3 are the conditions of the PCR reactions and the primer sequences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePNF regulated miRNAs in ALF, severe drug induced liver injury and fatty liver disease.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePNF regulated miRNAs of the present study\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIndependent confirmation of PNF regulated miRNAs in various pathological conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSerum / Tissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePubMed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003emiRNA 122-5p\u003c/p\u003e \u003cp\u003emiRNA 122-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eacute liver failure and spontaneous remission from ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;, T\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esevere drug induced liver injury (DILI) progressing to ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSteatosis, NASH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;, T\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 125b-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHBV-ACLF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eALF, Regulator of cell death\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esDILI / ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNASH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 192-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esDILI / ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNAFLD, NASH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 27b-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esDILI / ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSteatosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 103a-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esDILI / ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSteatosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 194-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esDILI / ALF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eWe compared PNF associated miRNAs to published findings for acute liver failure and severe drug induced liver injury (DILI) cases. Note the DILI cases are mainly due to acetaminophen overdose. S\u0026thinsp;=\u0026thinsp;Serum, T\u0026thinsp;=\u0026thinsp;Tissue, P\u0026thinsp;=\u0026thinsp;Plasma \u0026uarr;=upregulation, \u0026darr;=downregulation.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData-analysis\u003c/strong\u003e \u003cp\u003eWe applied the 2\u003csup\u003e\u0026minus;(∆∆CT)\u003c/sup\u003e method to calculate changes of disease regulated miRNAs using the following formula\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e∆CT\u0026thinsp;=\u0026thinsp;CT (miRNA of interest) \u0026ndash; CT (reference gene)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e∆∆CT = ∆CT (patient sample) \u0026ndash; ∆CT (healthy control)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eFold change\u0026thinsp;=\u0026thinsp;2^\u003csup\u003e-(∆∆CT)\u003c/sup\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eWe used RNU6B as a reference gene to determine the ∆Ct value of a miRNA of interest of either fresh or FFPE liver tissue material. In the case of plasma extracts the mean of miRNA16-5p and Cel-miR-39-3p served as a reference gene. To evaluate the regulation of miRNAs among PNF cases and to determine fold changes, we calculated the ∆CT values of N\u0026thinsp;=\u0026thinsp;11 individual controls and used the average ∆CT for comparisons with individual PNF cases. Additionally, we investigated the regulation of miRNAs in the circulation pre- and post-surgery (up to 3 days) and compared the results with tissue extracts of the same patients.\u003c/p\u003e \u003cp\u003eWe applied the Shapiro\u0026ndash;Wilk method to test for normality. Depending on the data distribution, we used the paired/unpaired t-test or the non-parametric Mann-Whitney-Test or Wilcoxon matched-pairs signed rank test. A * denotes a significant \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. We performed all statistical computations with the GraphPad Prism software version 8.4.3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSearch for miRNA gene targets\u003c/h2\u003e \u003cp\u003eBased on a fatty allograft PNF disease model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] we performed genome wide scan to identify target genes of significantly regulated miRNAs in PNF. Among the highly regulated miRNAs, we focused on those with an established role in hepatic lipid metabolism, liver injury and regeneration as well as programmed cell death. Based on genome wide miRNA scans, we selected 15 miRNAs that were highly regulated and performed gene ontology annotations to convert rat miRNAs into their human orthologues using the g-profiler program [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Subsequently, we queried the miRNet public repository to identify potential genes targeted by the selected miRNAs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We compared the list of potential targets with significantly regulated genes which we identified in fatty allograft failing livers of the rat (PNF study) and searched for common targets. This defined 2,307 genes, and we visualized the miRNA-target gene networks with the Cytoscape software (U.S. National Institute of General Medical Sciences, NIGMS). Additionally, we evaluated the biological functions of the regulated target genes with the gene ontology tool Metascape [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and David database [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and created visual networks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRat Serum\u003c/h2\u003e \u003cp\u003eDetails regarding the animal study are given in our recent publication (see Kulik et al., 2024 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]), and the study is reported in accordance with ARRIVE guidelines. Ethical approval was granted by the animal welfare ethics committee of the State of Lower Saxony, Germany (\u0026ldquo;Lower Saxony State office for Consumer Production and Food Safety\u0026rdquo; [LAVES]). The approval ID is Az: 33.14-42502-04-13/1258. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003cp\u003eBlood samples were obtained from (CTx) and fatty allograft recipients (MTx) post OLT on day 1, 3, 7 and 14. We prepared serum from whole blood using standard procedures and performed a genome wide search for regulated miRNA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This defined differentially expressed miRNAs in the circulation of fatty allograft recipient animals, and we selected 15 miRNAs for their time dependent regulation and clinical validation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRationale for miRNA selection\u003c/h2\u003e \u003cp\u003eBased on findings from a disease model of fatty allograft associated PNF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we selected 15 highly regulated miRNAs with known functions in the control of lipid metabolism, apoptosis, acute liver failure and liver regeneration (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eShown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e are H\u0026amp;E-stained liver sections of PNF allografts with varying degree of hepatic steatosis, inflammation and necrosis. Case A1 is a 43 year (y) old male who received a fatty allograft. Histology of the first allograft evidenced marked centrilobular and subcapsular map-like necrosis excessive inflammatory infiltrates, shrunken and vacuolated hepatocytes and macrovesicular steatosis (Case A1). Case A2 is a 58y old female. Note the marked macrovesicular steatosis, the centrilobular necrosis, the partial destruction of portal fields and extra-hepatic bile duct necrosis. Panel B1 and B2 refers to a 39y old female. Here, histology revealed subtotal necrosis indicative for excessive reperfusion injury. A further example relates to a 40y old female, and the liver section in C1 shows mixed micro- and macrovesicular steatosis, parenchymal necrosis and ischemia/reperfusion injury. Panel C2 refers to a 57y old male patient, and this liver section shows primarily macrovesicular steatosis, perivenular cholestasis with occasional lymphocytic infiltrates. Depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e panel D1 is the case of a 60y old male with marked portal inflammatory infiltrates, extensive lobular necrosis and macrovesicular steatosis of the fatty allograft. Case D2 refers to a 49y old female, and the allograft shows excessive macrovesicular steatosis, fresh hemorrhage, centrilobular necrosis and acute fatty liver dystrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePNF-associated miRNAs\u003c/h2\u003e \u003cp\u003eTo validate PNF associated miRNAs, we analyzed fifty-nine FFPE tissue blocks of 29 PNF cases and compared the data to 11 individual controls, i.e. morphological normal liver resection material obtained in the course of an elective hepatobiliary surgery (supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, we addressed the question whether the degree of hepatic steatosis influenced their regulation.\u003c/p\u003e \u003cp\u003eFirst, we considered the expression of the house keeping gene RNU 6B. Its expression did not differ between controls and PNF cases irrespective of the degree of hepatic steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Therefore, the selection of the housekeeping gene is justified and could be used as a \u0026ldquo;normalizer\u0026rdquo; in qPCR assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, we considered the interpatient variability in the expression of individual miRNAs and found all miRNAs to behave similar (supplementary Table S4). Therefore, we exclude sampling bias as a possible confounder.\u003c/p\u003e \u003cp\u003eThird, we computed the 2\u003csup\u003e\u0026minus;(∆∆CT)\u003c/sup\u003e-values of miRNAs among PNF cases and found 11 out of 15 miRNAs to be significantly regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026amp;C, non-regulated miRNAs in supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Except for miRNA-125a-5p and miRNA-195-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) the miRNAs were repressed in expression when compared to morphologically normal tissue as exemplified for miRNA-26a-5p and miRNA-27b-3p which were repressed to about 30% of controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Note, the latter two miRNAs were highly repressed in 80% and 85% of cases.\u003c/p\u003e \u003cp\u003eFourth, we addressed the question whether the degree of hepatic steatosis influenced expression of PNF-associated miRNAs. While for the majority of PNF regulated miRNAs the expression remained alike (supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB), we found miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p to be significantly influenced by the degree of hepatic steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The results suggest that hepatic steatosis aggravated the repression of these miRNAs.\u003c/p\u003e \u003cp\u003eFifth, we compared the regulation of PNF associated miRNAs in clinical samples to findings obtained from the animal study and the results were comparable (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For instance, in rat liver and human PNF cases miRNA-122-5p was repressed to 35% and 34% of controls. Notwithstanding, there are also significant differences in PNF associated miRNA regulations between human cases and the animal model. Specifically, let-7b-5p, miRNA-125a-5p, miRNA-126-3p, miRNA-194-5p and miRNA-195-5p were oppositely regulated between clinical cases and the animal model and the changes were more pronounced in the animal PNF-model.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eRegulation of PNF associated miRNAs in human and rat liver tissue.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePNF rat\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMean, 95%-CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePNF human\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMean, 95%-CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePNF human\u003c/span\u003e\u003c/p\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLet-7b-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15% (13\u0026ndash;18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e211% (100\u0026ndash;302%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-19b-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11% (10\u0026ndash;12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51% (41\u0026ndash;70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-23b-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4% (4\u0026ndash;5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65% (48\u0026ndash;89%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-26a-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6% (6\u0026ndash;7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% (24\u0026ndash;36%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-27b-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48% (25\u0026ndash;100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30% (21\u0026ndash;36%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-103a-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6% (6\u0026ndash;7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5% (43\u0026ndash;59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-122-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4% (4\u0026ndash;4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28% (17\u0026ndash;52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-122-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35% (29\u0026ndash;45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34% (30\u0026ndash;73%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-125a-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4% (4\u0026ndash;4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e166% (130\u0026ndash;215%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-125b-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3% (3\u0026ndash;3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53% (42\u0026ndash;66%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-126-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3% (3\u0026ndash;3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e154% (119\u0026ndash;195%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-192-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21% (18; 25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55% (35\u0026ndash;67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-194-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7% (6; 8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e105% (77\u0026ndash;137%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-195-5p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4% (4; 4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e228% (172\u0026ndash;301%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emiRNA-455-3p\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8% (7; 8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46% (33\u0026ndash;54%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eThe data are mean and 95%-CI-values.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIschemia injury\u003c/h2\u003e \u003cp\u003eTo assess the effects of ischemia-injury on the regulation of PNF associated miRNAs, we evaluated their expression in biopsy of liver allografts (N\u0026thinsp;=\u0026thinsp;7, supplementary Table S5A) prior to transplantation. Additionally, we obtained intraoperative biopsies after hilus occlusions during hepatic surgery (N\u0026thinsp;=\u0026thinsp;10, supplementary Table S5B) and compared the regulation of miRNAs of donor liver biopsies during organ storage to intra-operative liver biopsies taken from patients undergoing elective hepatobiliary surgery. The data shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e are ∆CT values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) for significantly upregulated miRNAs following hilus occlusion. For instance, miRNA-122-5p was nearly 3-fold upregulated in liver tissue following ischemia injury (median: 2.6-fold, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This miRNA is a well-known marker of liver injury. Independent research demonstrated miRNA-122-5p to be highly enriched in the nucleus of liver cells and to block activity of the cell survival oncomiR miR-21 at the posttranscriptional level [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Since ischemia injury is associated with marked cellular damage, its release into circulation is expected. Indeed, we found blood borne miRNA-122-5p to be 19-fold upregulated on day 1 post-surgery and this represents a\u0026thinsp;\u0026gt;\u0026thinsp;600% increase of this miRNA when compared to its induced tissue expression (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, tissue expression of let-7b-5p, miRNA-125b-5p and miRNA-194-5p were significantly upregulated by about 3-fold, and these miRNAs are known to augment inflammation and hepatic stellate cell activation. For comparison, the data of non-significantly regulated miRNAs are given in supplementary Figure S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003emiRNA in the systemic circulation following reperfusion injury of clinical cases\u003c/h2\u003e \u003cp\u003eIschemia/reperfusion (IR) injury is an unavoidable process in hepatic surgery and to determine whether the selected miRNA biomarkers are also regulated in response to IR injury, we investigated their regulation in blood samples taken prior to (T\u0026thinsp;=\u0026thinsp;0) and post-surgery on day 1, 2 and 3.\u003c/p\u003e \u003cp\u003eWe evaluated 14 patients of which one-half were OLT cases, and the other half consisted of elective surgeries for primary or secondary liver malignancies (supplementary Table S6). We obtained serial blood samples from the same patients and determined CT-values by the 2\u003csup\u003e\u0026minus;(∆∆\u0026minus;CT)\u003c/sup\u003e-method. We used the average CT-values of miRNA-16-5p and Cel-miRNA-39-3p as reference genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and independent research demonstrated the advantageous of applying the average of two reference genes for data analysis [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The time course of individual blood borne miRNAs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-G, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e out of 15 PNF associated miRNAs, i.e., 27b-3p, 122-3p,122-5p,125b-5p,192-5p and miRNA-194-5p were significantly upregulated in plasma by a range of 7- 26-fold post-surgery. Importantly, we observed opposite regulation of tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and blood borne miRNAs and this highlights their sensitivity to ischemia-reperfusion injury (IRI). All regulated miRNAs returned to pre-surgery or even below T\u0026thinsp;=\u0026thinsp;0 expression values on day 3 post-surgery and unchanged miRNAs are given in supplementary Figure S4. However, miRNA-126-3p remained consistently repressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) and experimental research demonstrated this miRNA to be a target Hoxb6. This transcription factor controls expression of SOX9 in liver progenitor cells which are destined to replace damage cells following CCL4 liver injury of mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, a regulatory loop exists between miRNA-126-3p, Hoxb6 and SOX9, and it is tempting to speculate that repressed miRNA-126-3p serum levels in clinical samples signify delayed liver regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we searched for blood borne miRNAs either regulated in OLT or tumor associated surgery. Depicted in supplementary Figure S5, panel A are miRNAs which are explicitly regulated in tumor liver resection cases, i.e. miRNA-19b-3p, miRNA-125a-5p and miRNA-126-3p and these function in wound repair and fibrosis, liver regeneration and metabolic disease. For instance, overexpression of miRNA-125a-5p supports liver regeneration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Conversely, miRNA-27b-3p and miRNA-194-5p are specifically regulated in OLT (supplementary Figure S5, panel B) and these function in inflammation and rejection of the graft [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate different grades of hepatic steatosis on the regulation of blood borne miRNA, we compared plasma samples of patients diagnosed with mild to moderate steatosis (N\u0026thinsp;=\u0026thinsp;7) to cases of marked steatosis (N\u0026thinsp;=\u0026thinsp;7). Obviously, the number of patients is small, but even so, miRNA-103a-3p reached statistical significance and this miRNA promotes hepatic steatosis by repressing the expression of palmitoyl-CoA oxidase [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, the expression of miRNA-26a-5p, miRNA-27b-3p, miRNA-103a-3p and miRNA-122-5p tended to be higher in cases of marked steatosis (supplementary Figure S6), however, did not reach statistical significance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSerum miRNAs in a rat fatty allograft OLT model\u003c/h2\u003e \u003cp\u003eWe recently reported the development of a PNF fatty allograft disease model [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]and determined the regulation of 15 miRNAs in blood samples of rats following liver transplantation on days 1, 3, 7 and 14 post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We compared their expression in serum of non-transplanted Chow-fed controls to serum values following OLT of healthy allografts. The data are shown as fold changes and we observed upregulation of miRNA-27b-3p, miRNA-122-3p, miRNA-122-5p, miRNA-125a-5p, miRNA-126-3p, miRNA-192-5p, miRNA-194-5p and miRNA-195-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, range 25 to 2-fold). Conversely, miRNA-7b-5p, miRNA-19b-3p, miRNA-23b-3p, miRNA-26a-5p, miRNA-125b-5p and miRNA-455-3p were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, range 22 to 1.5-fold). Although the latter miRNAs were below the expression of Chow-fed controls, it is obvious that their expression increased with time, thus implying improved liver function following OLT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we compared the regulation of PNF associated serum miRNAs in donor animals on a CHOW and MCD diet for 7 and 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e, left panels). Essentially, we observed mild increases of these miRNAs (range 2 to 4-fold) in MCD fed animals. This demonstrates their fatty liver associated regulation.\u003c/p\u003e \u003cp\u003eSubsequently, we compared the regulation of PNF associated miRNAs in serum of rats following OLT of healthy and fatty allografts. Shown in the right panels of Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e are serum miRNAs regulated in recipient animals following OLT of fatty allografts. The data are fold changes by comparing it to healthy allografts, and we divided the results into up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and down regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) miRNAs post OLT for up to 7 days. We observed marked induction of miRNA-122-3p and miRNA-122-5p, i.e. 48- and 10-fold induced, respectively in fatty allografts on day 7 post-OLT. Similarly, we observed a 19-fold increased expression of miRNA-194-5p in fatty allografts on day 7 post OLT. Furthermore, miRNA-126-3p, miRNA-192-5p and miRNA-194-5p were upregulated on day 1, 3 and 7 but downregulated on day 14 post-OLT.\u003c/p\u003e \u003cp\u003eConversely, Let-7b-5p, miRNA-27b-3p, miRNA-103-3p, miRNA-125a-5p, miRNA-125b-5p were down regulated in recipient animals of fatty allografts. Meanwhile, miRNA-19b-3p, miRNA-23b-3p and miRNA-26a-5p were unchanged up to 7 days post-OLT but declined thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eExcept for miRNA-122, all serum miRNAs were significantly down regulated on day 14 post-OLT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Thus, fatty allograft OLTs are hallmarked by repression of PNF associated miRNAs and the results are similar to clinical samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) on day 14 post-OLT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003emiRNA gene-target networks in PNF\u003c/h2\u003e \u003cp\u003eWe performed a genome wide scan to identify genes regulated in PNF cases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and for this purpose compared the transcriptomes of rat liver following OLT of healthy donor allografts (CTx) to PNF cases. This revealed 2450 differential expressed genes (DEGs) of which 2215 or nearly 91% were repressed. Therefore, PNF caused an unprecedented repression of the transcriptome and involved various components of the general transcription machinery including the CAAT enhancer binding proteins, TATA-Box binding protein associated factors, i.e. TAF-proteins, and various liver enriched transcription factors (HNF4alpha and FOXA3, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Strikingly, the BRD4 bromodomain and extra-terminal domain transcriptional activator is highly induced (\u0026gt;\u0026thinsp;10-fold) and was recently shown to be key player in the global loss of activity of the transcriptional machinery in damaged livers [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Typically, BRD4 binds to acetylated lysine residues of the chromatin (super-enhancers) and supports transcriptional activation of genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we queried the miRnet public data base to search for experimentally proven targets of 15 PNF associated miRNAs and compared the results to DEGs identified in PNF livers. We focused on target genes coding for hepatic lipid metabolism, liver injury and regeneration as well as programmed cell death and report results for 363 target genes. Depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e are the network for autophagy (panel 7B), apoptotic signaling (panel 7C), response to endoplasmic reticulum stress (panel 7D), response to hypoxia (panel 7E), mitochondrial organization (panel 7F) and response to cytokine (panel 7G). Note, the majority of target genes are repressed, and 4/5 of the regulated genes are experimentally proven targets while the remaining are predicted targets.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on findings from a preclinical liver transplant model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we aimed at validating fatty allografts associated miRNAs predictive for PNF. We confirmed clinical significance for 11 miRNAs, of which 9 and 2, respectively were down and upregulated (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our study revealed the significant relationship between the degree of hepatic steatosis and the repression of miRNA-27b-3p, miRNA-122-3p, miRNA-125b-5p and miRNA-192-5p in liver tissue of clinical PNF-cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Note under hypoxic conditions, and through upregulation of the transcription factor PPARγ, miRNA-27b plays an essential role in lipid metabolism [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] while silencing of miRNA-125b-5p promotes liver fibrosis in nonalcoholic fatty liver disease via integrin α8-mediated activation of the RhoA signaling pathway [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, repressed miRNA-192-5p aggravates lipid deposition by controlling the expression of stearoyl-CoA desaturase 1 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, we explored the regulation of PNF associated miRNAs in liver biopsies taken prior to OLT and intraoperatively following hilus occlusion. We found 4 miRNAs (let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p) significantly upregulated when biopsies following hilus occlusion were compared to T0 liver biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, we identified 6 up- and 1 downregulated miRNA in post-surgery blood samples of successfully performed OLTs and tumor liver resection cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Importantly, these miRNAs were oppositely regulated when compared to PNF cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, we demonstrate selectivity and specificity and clinical relevance for the majority of the miRNAs. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the 15 miRNAs and their regulation in FFPE-PNF-tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), pre- and intraoperative liver biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and blood samples taken from patients which underwent elective hepatobiliary surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eRegulation of miRNAs in human fatty allograft associated PNF cases, in T0 liver biopsies of healthy allografts prior to OLT and hepatectomies of tumor resection.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003emiRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eTissue expression in fatty allograft associated PNF\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eOLT healthy allografts and hepatectomy of neoplasms\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasma\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLiver tissue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 122-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; **\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr; *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 125b-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; **\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr; ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 27b-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; ****\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 122-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; ***\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 192-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; **\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 26a-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; ****\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 23b-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; *\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 103a-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; **\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 455-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; *\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 125a-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026uarr; *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 195-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026uarr; *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 194-5p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr; **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr; *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 126-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026darr; *\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLet 7b-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr; **\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiRNA 19b-3p\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we summarize the various functions of PNF associated miRNAs in the control of lipid metabolism, acute liver failure, IR-injury and liver regeneration. The regulation of miRNA-122 and its two mature products, i.e. miRNA-122-3p and \u0026minus;\u0026thinsp;5p is an interesting example [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although abundantly expressed in the liver, miRNA-122-3p is not significantly regulated in liver tissue resection material (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); however, is highly upregulated in blood samples following surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). On the first day post-surgery, its regulation ranged between 0.7 and 270-fold across individual patients and this miRNA serves as a marker of liver cell damage. In contrast, miRNA-122-5p is mildly but significantly upregulated in intra-operative biopsy samples following hilus occlusion (median\u0026thinsp;=\u0026thinsp;2.6-fold) and markedly increased in blood samples of the same patients (median\u0026thinsp;=\u0026thinsp;20-fold). miRNA-122 is essential for liver metabolic homeostasis and lipid metabolism. It exerts anti-inflammatory and anti-fibrotic properties and blocks viral replication in hepatocytes [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notwithstanding one report suggests liver injury-induced release of miRNA-122 to stimulate pulmonary inflammation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Typically, its expression is low in serum but highly upregulated during liver injury. Interestingly, in patients with spontaneous recovery from acute liver failure miRNA-122 is significantly upregulated in serum and liver tissue when compared to no recovered patients. This implies an important role of this miRNA in instructing liver regeneration [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA further example relates to miRNA-125b-5p which was reported to alleviate acute liver failure by regulating the Keap1/Nrf2/HO-1 pathway [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, this miRNA protects from reperfusion injury by inhibiting TRAF6 and NF-κB signaling [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnlike preclinical PNF cases, let-7b-5p is regulated in human liver tissue, but not in blood samples, and this miRNA inhibits cell proliferation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Notwithstanding, one study identified repressed let-7b blood levels in children diagnosed with progressive familial intrahepatic cholestasis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In vitro, this miRNA inhibits hepatic stellate cell activation and therefore plays a role in fibrosis [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA recent review summarized significantly regulated miRNAs in human acute liver failure (ALF) cases [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The review is based on 21 independent studies and primarily describes findings for acetaminophen overdose and drug induced liver injury (DILI) cases as well as viral liver disease. Of the ALF serum and plasma regulated miRNAs, 53% are common to our study, i.e. 8/15 miRNA, and this demonstrates relevance of these miRNAs in acute liver failure across independent clinical studies. Although the causes of ALF and PNF are different in nature, i.e. drug induced versus fatty allograft associated PNFs, the results underscore the clinical relevance of the selected miRNAs and their utility as commonly regulated biomarkers in PNF and ALF.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e compiles miRNAs commonly regulated between clinical ALF and fatty allograft associated PNF cases and highlights their basic function in liver biology. For instance, miRNA-27b-3p regulates mitochondrial biogenesis [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and targets several key lipid-metabolism genes [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This miRNA is highly repressed in fatty allograft associated PNF cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and given its role in mitochondrial biogenesis, its repression might be regarded as an adaptive response. Indeed, an inverse relationship exists between miRNA-27b expression and mitochondria content [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Similarly, de novo lipogenesis can be inhibited by miRNA-27a. This miRNA alleviates obesity-initiated NAFLD by repressing the expression of fatty acid synthase and stearoyl-CoA desaturase [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Furthermore, miRNA-27b-5p inhibits PPARγ driven lipogenesis [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA further example relates to an identification of circulating miRNAs in NAFLD patients. Specifically, Pirola and co-workers investigated serum microRNAs among liver biopsy proven NAFLD cases and healthy controls [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Of the 84 investigated miRNAs, blood borne miRNA-122, miRNA-192, miRNA-19a and miRNA-19b, miRNA-125b proved to be of diagnostic value. In the present study miRNA-122-5p, miRNA-192-5p, miRNA-125b-5p were highly significantly repressed among fatty allograft associated PNF cases and their regulation was influenced by the hepatic lipid content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Unlike liver biopsy and serum findings for NAFLD patients [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] miRNA-19b-3p was not significantly regulated in fatty allograft associated PNF cases (supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, panel A).\u003c/p\u003e \u003cp\u003eThe role of miRNA-192-5p in human diseases is the subject of a recent review and there is evidence for this miRNA to effect energy metabolism [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Downregulation of miRNA 192 causes hepatic steatosis through upregulation of sterol regulatory element binding transcription factor 1 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In the present study, miRNA-192-5p was markedly repressed among fatty allograft associated PNF cases. Its regulation was influenced by the hepatic lipid content (Fig.\u0026nbsp;2D5) and correlated with the degree of steatosis. Conversely, miRNA-192-5p is significantly upregulated in blood samples of patients following OLT of healthy allografts and patients undergoing hepatectomy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, the importance of the HNF4α-miRNA-194/192 signaling axis in maintaining hepatic cell function was demonstrated in liver-specific Hnf4a-null (Hnf4aΔH) mice [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and miRNA-192-5p and miRNA-194-5p are localized in a cluster. Note both miRNAs were significantly upregulated in plasma samples following hepatic surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and this demonstrates its diagnostic relevance for distinguishing PNF from liver regeneration cases.\u003c/p\u003e \u003cp\u003eAnother miRNA linked to liver regeneration is miRNA-26a. This miRNA is significantly repressed in PNF cases but abundantly expressed in OLT biopsy and blood samples of patients undergoing elective liver surgery (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Independent research demonstrated that the growth factor termed augmenter of liver regeneration (ALR) induces expression of miRNA 26a and stimulated cell proliferation via the microRNA-26a/Akt/cyclin D1 signaling pathway [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Conversely, miRNA-26 influences the cross-talk between mdm2 and p53 and its repression stimulates mdm2 expression which inhibits p53 activity [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Another study demonstrated down-regulation of microRNA-26a to promote mouse hepatocyte proliferation during liver regeneration [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Therefore, repressed miRNA-26 supports liver regeneration and can be regarded as an adaptive response to impair programmed cell death.\u003c/p\u003e \u003cp\u003eLiver regeneration is supported by the upregulation of miRNA-125a-5p, and its overexpression in the human liver cell line HL-7702 increased cell viability significantly [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In the present study, miRNA-125a-5p was one of the two significantly increased miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and we consider its upregulation in fatty allograft associated PNF cases as an attempt to stimulate liver regeneration. Notwithstanding, miRNA-195-5p was also significantly upregulated and this miRNA promotes hepatic stellate cell activation and liver fibrosis by suppressing PTEN expression in a mouse model of liver damage [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Furthermore, down-regulation of miR-23b stimulated TGF-β1/Smad3 signaling during the termination stage of liver regeneration [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] and therefore contributes to impaired liver regeneration. Consistent with its function miR-23b is repressed in fatty allograft associated PNF cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLastly, we observed repressed plasma miRNA-126-3p in post-surgery blood samples of OLTs and tumor liver resection cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This miRNA suppresses inflammation in endothelial cells [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], is significantly repressed in higher grade NAFLD patients [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], and its repression impairs liver regeneration in mice following partial hepatectomy [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on their specific regulation by the grade of hepatic steatosis, we propose miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p as a panel of diagnostic miRNAs to predict fatty allograft associated PNF. Their validation in prospective clinical trials is warranted. In addition, miRNA-26a-5p is highly regulated in most PNF cases (80%) and therefore is a biomarker candidate worthwhile for in depth validation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe report an identification of miRNAs significantly associated with fatty allograft associated PNFs. Our findings warrant clinical validation to demonstrate their prognostic value.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitations\u003c/h2\u003e \u003cp\u003eWe would like to highlight the following study limitations. First, PNF is a rare and unpredictable event, and therefore it is difficult to design a prospective study. Thus, our findings are based on archived tissue materials. Second, given the retrospective nature of the study, we are unable to control bias, i.e. the outcome was known prior to study initiation. However, we do not consider this bias to be of critical importance for an identification of PNF biomarkers. Third, we were unable to obtain sufficient number of intraoperative liver biopsies following OLT of healthy allograft. Fourth, although we included all fatty allograft associated PNF cases among 1,200 OLTs performed at our institution, we report a single center study. Nonetheless, the power analysis showed the number of cases to be sufficient to determine statistical significance. Future studies should be based on randomized clinical trials.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eALF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacute liver failure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eATP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCCC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echolangiocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ecDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomplementary DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCLM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecolorectal liver metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecrossing threshold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDEGs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edifferential expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDILI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edrug induced liver injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edesoxyribonuclein acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEAD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eearly allograft dysfunction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eECD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextended criteria donors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEDTA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethylene diamine tetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eER\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendoplasmatic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEtOH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFFPE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eformalin-fixed and paraffin-embedded tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH\u0026amp;E\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin and eosin staining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHCC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIRI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eischemia-reperfusion injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTx\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enormal liver recipient rats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMCD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethionine/choline deficient diet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emiRNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicro ribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMTx\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty liver recipient animals\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNAFLD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-alcoholic fatty liver disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNASH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-alcoholic steatohepatitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOLT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eorthotopic liver transplantation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePNF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprimary non-function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eqPCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRpm\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erounds per minute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereverse transcription\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTCA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etricarboxylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eY\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eyears\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained approval from the Ethics Committee of the Hannover Medical School (8582_BO_S_2019, 8368_Bo_K_2019 and 7506_Bo_K_2017) for the use of archived PNF tissue blocks, fresh liver resection material and blood samples from patients\u0026nbsp;undergoing elective hepatobiliary surgery. Informed consent was obtained from all subjects and/or their legal guardian(s). All methods were performed in accordance with the relevant guidelines and regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetails regarding the animal study are given in our recent publication (see Kulik et al., 2024 \u003cspan lang=\"EN-GB\"\u003e[14]\u003c/span\u003e), and the study is reported in accordance with ARRIVE guidelines. Ethical approval was granted by the animal welfare ethics committee of the State of Lower Saxony, Germany (\u0026ldquo;Lower Saxony State office for Consumer Production and Food Safety\u0026rdquo; [LAVES]). The approval ID is Az: 33.14-42502-04-13/1258. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLower Saxony Ministry of Culture and Sciences and the Volkswagen Foundation, Germany to JB (25A.5-7251-99-3/00).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJS collected the samples and performed miRNA analysis and qPCR measurements. JB performed the histopathology study. Both authors analyzed the data. JS prepared the figures and supported the writing of the manuscript. JB wrote the final manuscript. Both authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Ulf Kulik and the Clinic for General, Visceral and Transplant Surgery of Hannover Medical School for providing biopsies and blood samples and the Institute of Pathology for supplying FFPE tissue blocks of PNF cases. We gratefully acknowledge the technical support of Gabi Onken in the histopathology work.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLock JF, Schwabauer E, Martus P, Videv N, Pratschke J, Malinowski M, Neuhaus P, Stockmann M (2010) Early diagnosis of primary nonfunction and indication for reoperation after liver transplantation. Liver Transpl 16:172\u0026ndash;180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClavien PA, Camargo CA, Croxford R, Langer B, Levy GA, Greig PD (1994) Definition and classification of negative outcomes in solid organ transplantation. Application in liver transplantation. Ann Surg 220:109\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulik U, Lehner F, Klempnauer J, Borlak J (2017) Primary non-function is frequently associated with fatty liver allografts and high mortality after re-transplantation. Liver Int 37:1219\u0026ndash;1228\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYounossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M (2016) Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 64:73\u0026ndash;84\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahini N, Borlak J (2014) Recent insights into the molecular pathophysiology of lipid droplet formation in hepatocytes. Prog Lipid Res 54:86\u0026ndash;112\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBedossa P (2017) Pathology of non-alcoholic fatty liver disease. Liver Int 37(Suppl 1):85\u0026ndash;89\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunt EM, Tiniakos DG (2010) Histopathology of nonalcoholic fatty liver disease. World J Gastroenterol 16:5286\u0026ndash;5296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKleiner DE, Brunt EM (2012) Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research. Semin Liver Dis 32:3\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyvazoglu Soy EH, Boyvat F, Ozdemir BH, Haberal N, Hilmioglu F, Haberal M (2018) Liver Biopsy Results in Potential Donor Evaluation in Living Related Liver Transplant. Exp Clin Transpl 16(Suppl 1):35\u0026ndash;37\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNemes B, Gaman G, Polak WG, Gelley F, Hara T, Ono S, Baimakhanov Z, Piros L, Eguchi S (2016) Extended-criteria donors in liver transplantation Part II: reviewing the impact of extended-criteria donors on the complications and outcomes of liver transplantation. Expert Rev Gastroenterol Hepatol 10:841\u0026ndash;859\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVodkin I, Kuo A (2017) Extended Criteria Donors in Liver Transplantation. 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Physiol Rep 4:e12661. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.14814/phy2.12661\u003c/span\u003e\u003cspan address=\"10.14814/phy2.12661\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"liver failure, ischemia injury, non-coding RNA","lastPublishedDoi":"10.21203/rs.3.rs-4616493/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4616493/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Primary non-function (PNF) of an allograft defines an irreversible graft failure and although rare, constitutes a life-threatening condition that requires high-urgency re-transplantation. Equally, drug induced acute liver failures (ALF) are seldom but the rapid loss of hepatic function may require orthotropic liver transplantation (OLT). Recently, we reported the development of a PNF-disease model of fatty allografts and showed that a dysfunctional Cori and Krebs cycle and inhibition of lactate transporters constitute a mechanism of PNF. We identified highly regulated miRNAs and their target genes and selected 15 miRNA-biomarker candidates for clinical validation. Our study aimed at their clinical validation. Additionally, we assessed their diagnostic value in ALF. We performed RT-qPCRs of 15 miRNA-biomarker candidates in well-documented PNF cases following OLT of fatty allografts. To assess specificity and selectivity, we compared their regulation in pre- and intraoperative liver biopsies and post-operative in blood samples of patients undergoing elective hepatobiliary surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe confirmed regulation of 11 PNF-associated miRNAs in clinical PNF cases and found expression of miRNA-27b-3p, miRNA-122-3p, miRNA-125a-5p, miRNA-125b-5p and miRNA-192-5p to correlate with the hepatic steatosis grade. Furthermore, we demonstrate selectivity and specificity for the biomarker candidates with opposite regulation of let-7b-5p, miRNA-122-5p, miRNA-125b-5p and miRNA-194-5p in blood samples of patients following successful OLTs and/or liver resection. Strikingly, and based on 21 independent studies, eight PNF-associated miRNAs are also regulated in ALF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e We report miRNAs highly regulated in PNF and ALF. Their common regulation in different diseases broadens the perspective as biomarker candidates for an identification of patients at risk for PNF and ALF.\u003c/p\u003e","manuscriptTitle":"miRNA biomarkers to predict risk of primary non-function of fatty allografts and drug induced acute liver failures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 20:11:08","doi":"10.21203/rs.3.rs-4616493/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-11T08:01:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-31T10:43:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52191694866982204386925385820625891347","date":"2024-08-26T06:30:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-02T14:31:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-23T16:12:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T14:17:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2024-06-21T09:40:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a28b315a-4c64-4ade-9734-1d82df143059","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:05:32+00:00","versionOfRecord":{"articleIdentity":"rs-4616493","link":"https://doi.org/10.1007/s11010-024-05129-3","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2024-10-18 15:57:02","publishedOnDateReadable":"October 18th, 2024"},"versionCreatedAt":"2024-07-18 20:11:08","video":"","vorDoi":"10.1007/s11010-024-05129-3","vorDoiUrl":"https://doi.org/10.1007/s11010-024-05129-3","workflowStages":[]},"version":"v1","identity":"rs-4616493","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4616493","identity":"rs-4616493","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}