Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

A Novel Prognostic Model of Hepatocellular Carcinoma per Two NAD+ Metabolic Synthesis-Associated Genes.

Hepatocellular carcinoma (HCC) is a formidable challenge to global human health, while recent years have witnessed the important role of NAD+ in tumorigenesis and progression. However, the expression pattern and prognostic value of NAD+ in HCC still remain elusive. Gene expression files and corresponding clinical pathological files associated with HCC were obtained from the Cancer Genome Atlas (TCGA) database, and genes associated with NAD+ were retrieved from the GSEA and differentially analyzed in tumor and normal tissues. A consensus clustering analysis was conducted by breaking down TCGA patients into four distinct groups, while Kaplan-Meier curves were generated to investigate the disparity in clinical pathology and endurance between clusters. A prognostic model based on NAD+-associated genes was established and assessed by combining LASSO-Cox regression, uni- and multi-variate Cox regression, and ROC curve analyses. Investigations were conducted to determine the expression of distinct mRNAs and proteins in both HCC and non-tumor tissues. A novel two-gene signature including poly (ADP-Ribose) polymerase 2 (PARP2) and sirtuin 6 (SIRT6) was obtained through LASSO-Cox regression and was identified to have favorable prognostic performance in HCC patients from TCGA. Analyses of both single and multiple variables showed that the prognostic model was a distinct prognostic factor in the endurance of liver cancer patients in both the training and trial groups. The nomogram also exhibited clinical significance in the prognosis of HCC patients. Immunohistochemistry, qRT-PCR, and Western blotting revealed that HCC samples exhibited higher PARP2 and SIRT6 expression levels than those of normal controls. This study identified a robust prognostic model comprising two NAD+-associated genes using bioinformatic methods, which is accurate in predicting the survival outcome of HCC patients. This model might benefit the early diagnosis of HCC and further facilitate the management of individualized medical service and clinical decision-making.

Transcriptome sequencing and metabolome analysis reveal the metabolic reprogramming of partial hepatectomy and extended hepatectomy.

Surgical resection remains a critical treatment option for many patients with primary and secondary hepatic neoplasms. Extended hepatectomy (eHx) may be required for some patients with large tumors, which may cause liver failure and death. Partial hepatectomy (pHx) and eHx mouse models were constructed, liver tissues were sampled at 18, 36, and 72 h posthepatectomy. Transcriptome and metabolome analyses were employed to explore the different potential mechanisms in regeneration and injury between pHx and eHx. The results showed that eHx was associated with more severe liver injury and lower survival rates than pHx. Transcriptomics data showed there were 1842, 2129, and 1277 differentially expressed genes (DEGs) in eHx and 962, 1305, and 732 DEGs in pHx at 18, 36, and 72 h posthepatectomy, respectively, compared with the those in the sham groups. Compared with pHx, the number of DEGs in the eHx group reached a maximum of 230 at 18 h after surgery and decreased sequentially to 87 and 43 at 36 and 72 h. Metabolomics analysis identified a total of 1399 metabolites, and 48 significant differentially produced metabolites (DPMs) were screened between eHx and pHx. Combined analysis of DEGs and DPMs indicated that cholesterol metabolism and insulin resistance may be two important pathways for liver regeneration and mouse survival postextended hepatectomy. Our results showed the global influence of pHx and eHx on the transcriptome and metabolome in mouse liver, and revealed cholesterol metabolism and insulin resistance pathways might be involved in regeneration post-pHx and -eHx.

Complement inhibition alleviates donor brain death-induced liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase signaling.

Brain death (BD) donors are the primary source of donor organs for liver transplantation. However, the effects of BD on donor livers and outcomes after liver transplantation remain unclear. Here, we explored the role of complement and the therapeutic effect of complement inhibition in BD-induced liver injury and posttransplantation injury in a mouse BD and liver transplantation model. For complement inhibition, we used complement receptor 2 (CR2)-Crry, a murine inhibitor of C3 activation that specifically targets sites of complement activation. In the mouse model, BD resulted in complement activation and liver injury in donor livers and a cascade liver injury posttransplantation, mediated in part through the C3a-C3aR (C3a receptor) signaling pathway, which was ameliorated by treatment with CR2-Crry. Treatment of BD donors with CR2-Crry improved graft survival, which was further improved when recipients received an additional dose of CR2-Crry posttransplantation. Mechanistically, we determined that complement inhibition alleviated BD-induced donor liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase (PI3K) signaling pathways. Together, BD induced donor liver injury and cascade injury post-transplantation, which was mediated by complement activation products acting on PI3K signaling pathways. Our study provides an experimental basis for developing strategies to improve the survival of BD donor grafts in liver transplantation.

Serine-threonine Kinase Receptor-Associated Protein is a Critical Mediator of APC Mutation-Induced Intestinal Tumorigenesis Through a Feed-Forward Mechanism.

BACKGROUND & AIMS: Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation-induced intestinal tumor initiation and progression. METHODS: We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor-derived organoids for STRAP knockdown and knockout and overexpression experiments. RESULTS: Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/ß-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/ß-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/ß-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the ß-catenin/TCF4 complex on the Strap promoter. CONCLUSIONS: STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-ß-catenin/STRAP regulatory axis.

DLK1-directed chimeric antigen receptor T-cell therapy for hepatocellular carcinoma.

BACKGROUND: Delta-like homologue 1 (DLK1), a transmembrane protein, is highly expressed in hepatocellular carcinoma (HCC). We explored whether DLK1-directed chimeric antigen receptor (CAR) T cells can specifically eliminate DLK1-positive HCC cells and serve as a therapeutic strategy for HCC immunotherapy. METHODS: We first characterized a homemade anti-human DLK1 monoclonal antibody, sequenced the single-chain Fragment variable (scFv) and integrated it into the second-generation CAR lentiviral vector, and then developed the DLK1-directed CAR-T cells. The cytotoxic activities of DLK1-directed CAR-T cells against different HCC cells were evaluated in vitro and in vivo. RESULTS: The genetically modified human T cells with the DLK1-directed CARs produced cytotoxic activity against DLK1-positive HCC cells. Additionally, the DLK1-directed CARs enhanced T cell proliferation and activation in a DLK1-dependent manner. Interestingly, the DLK1-targeted CAR-T cells significantly inhibited both subcutaneous and peritoneal xenograft tumours derived from human liver cancer cell lines HepG2 or Huh-7. CONCLUSION: DLK1-directed CAR-T cells specifically suppresses DLK1-positive HCC cells in vitro and in vivo. This study provides a novel transmembrane antigen DLK1 as a potential therapeutic target appropriate for CAR-T cell therapy, which may be further developed as a clinical therapeutic strategy for HCC immunotherapy.

Callispheres® drug-eluting beads transarterial chemoembolization might be an efficient and safety down-staging therapy in unresectable liver cancer patients.

PURPOSE: The purpose was to explore the effect of drug-eluting beads transarterial chemoembolization (DEB-TACE) on down-staging in unresectable liver cancer patients. METHODS: A total of 180 patients with PHC treated by TACE were retrospectively analyzed. These included 80 cases in the DEB-TACE group and 100 cases in the cTACE group. Of these, 56 had complete clinical data (DEB-TACE: 24, cTACE: 32), and 23 patients received hepatectomy after TACE as a down-staging therapy (DEB-TACE: 15, cTACE: 8). Data (including clinical characteristics, clinical efficacy, tumor response, tumor diameters, residual liver volume, and liver function indexes before and after TACE, RFS, OS, and complications were collected and compared. Treatment response was evaluated at 1 month after TACE. Tumor diameter was evaluated by abdominal computed tomography scan. The residual liver volume was evaluated by IQQA liver system, and relapse-free survival (RFS) and overall survival (OS) were calculated by Kaplan-Meier curves. RESULTS: The conversion rate in DEB-TACE group was higher than cTACE group (18.8% vs 8%, p = 0.032). In DEB-TACE group, 17 patients achieved objective response rate (ORR) which was higher than cTACE group (70.8% vs 34.4%, p = 0.007). The tumor necrosis rate was higher in DEB-TACE group, but there was no significant difference between the two groups (p = 0.053). Tumor diameter was decreased after TACE compared to before TACE (DEB-TACE: 9.4 ± 3.3 vs. 5.4 ± 3.5 cm, p = 0.003; cTACE: 9.7 ± 2.6 vs. 6.9 ± 2.2, p = 0.036). As to residual liver volume, it was increased after TACE compared to before TACE (1066.2 cm3 vs. 1180.3 cm3, p = 0.007) in DEB-TACE group, while there was no significant difference in cTACE group (1046.4 cm3 vs. 1170 cm3, p = 0.339) compared by paired-sample t-test, but there was no significant difference before and after TACE when compared by unpaired-sample t-test (p > 0.05). After TACE at 1 month, the AFP level in the DEB-TACE group was significantly lower than that in the cTACE group (p = 0.003). For survival, the median RFS was 26.0 months in DEB-TACE group and 15 months in cTACE group; there was significant difference between the two groups (p = 0.0465). As to OS, the median OS in DEB-TACE group was higher than that in cTACE group, but there was no significant difference between the two groups (p = 0.165). For safety profiles, in terms of liver function and adverse events, there was no significant difference between the two groups. CONCLUSION: Compared with cTACE, DEB-TACE might be a more efficient and safety down-staging treatment in unresectable liver cancer patients.

Comparison of surgical outcomes among open, laparoscopic, and robotic pancreatoduodenectomy: a single-center retrospective study.

BACKGROUND: There is no general consensus on the feasibility and safety of robotic pancreatoduodenectomy (RPD) and whether it increases surgical risks. The purpose of this study was to assess the safety, feasibility, and rationality of RPD by comparing perioperative data among open pancreatoduodenectomy (OPD), laparoscopic pancreatoduodenectomy (LPD), and RPD performed in our center in recent years. METHODS: Clinical data of patients had undergone RPD (n = 32), LPD (n = 21), and OPD (n = 86) in The First Affiliated Hospital of Guangxi Medical University between January 2016 and June 2020 were retrospectively collected and analyzed. RESULTS: RPD required more time for operation (537.2 min vs. 441.5 min, p < 0.001) than OPD did, but less time to remove abdominal drainage tube (12.5 d vs. 17.3 d, p = 0.001). The differences between the RPD group and LPD group were interesting, as the two groups had similar operation time (537.2 min vs. 592.9 min, p = 1.000) and blood loss (482.8 ml vs. 559.5 ml, p > 0.05), but the RPD group had a higher activity of daily living score on postoperative day 3 (35.8 vs. 25.7, p = 0.0017) and a lower rate of conversion to OPD (6.5% vs. 38.1%, p = 0.011). Regarding complications, such as the postoperative pancreatic fistula, abdominal hemorrhage, intra-abdominal infection, bile leakage, reoperation, and perioperative mortality, there were no significant differences among the three groups. CONCLUSIONS: Not only is RPD feasible and reliable, it also offers significant advantages in that it improves postoperative recovery of skills needed for everyday life, has a low conversion rate to open surgery, and does not increase surgical risks.

Application of da Vinci robot and laparoscopy on repeat hepatocellular carcinoma.

Background: Repeat laparoscopic liver resection has been used safely and effectively on hepatocellular carcinoma (HCC). However, few studies have been performed on repeat HCC surgery by a da Vinci robot. This study aims to evaluate the outcomes of the patients with repeat HCC treated using a da Vinci robot or laparoscopic system at a single centre. Methods: All of the patients with repeat HCC treated using a da Vinci robotic or laparoscopic system between April 2017 and April 2020 were included in this retrospective study. Results: There were 24 patients with a mean age of 56 years who underwent da Vinci robotic or laparoscopic surgery for treatment of repeat HCC who were included in this study. The operations lasted 152 ± 25 min and 142 ± 34 min. The average intraoperative blood loss was 284 ± 89 ml and 251 ± 92 ml. The average hospitalisation stay lasted 9 ± 2 days and 9 ± 3 days. The rates at which surgeons switched to open surgery were 9% and 23%. No serious perioperative or post-operative complications were encountered. Conclusion: Da Vinci robots can provide a precise dissection of the tissue under a perfect view. It is a technically feasible procedure for less rates at which surgeons switched to open surgery on repeat HCC.

Tetramethylpyrazine ameliorates hepatic fibrosis through autophagy-mediated inflammation.

BACKGROUND: Imbalanced immune response and hepatic fibrosis are key factors related to the progression of chronic liver diseases. Tetramethylpyrazine (TMP), a natural alkaloid, has been widely used for treating liver injury. In this study, we explored the effect of TMP on hepatic fibrosis and the related mechanisms regulating autophagy. METHODS: A rat model of hepatic fibrosis and a model using an hepatic stellate cell line (HSC-T6) were created using CCl4 and platelet-derived growth factor (PDGF). Staining with haematoxylin and eosin (HE), Masson's stain, and TUNEL were performed for pathological diagnosis. ELISA, Western blotting, and immunofluorescence analyses were conducted to determine the expression levels of the specific markers for fibrosis, autophagy, inflammation, and signalling pathways. RESULTS: TMP treatment significantly rescued pathological injury and hepatic fibrosis. It also alleviated imbalances in the immune system, accumulation of extracellular matrix, and autophagy signals in hepatic fibrosis. At the same time, we found that application of the autophagy inducer rapamycin enhanced the therapeutic effect of TMP, whereas the autophagy inhibitor 3-methyladenine, PI3K pathway inhibitor LY294002, and AKT pathway agonist SC79 did the opposite. CONCLUSIONS: TMP exerts therapeutic effects in hepatic fibrosis mainly through promoting autophagy to ameliorate inflammation by inhibiting the AKT-mTOR signalling pathway, providing a new perspective for the treatment of chronic liver diseases.

MicroRNA-329-mediated PTTG1 downregulation inactivates the MAPK signaling pathway to suppress cell proliferation and tumor growth in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a severe malignancy usually producing a poor prognosis and high mortality rate. MicroRNAs (miRNAs) have been reported in association with CCA; however, the role miR-329 plays in the CCA condition still remains unclear. Therefore, this study was conducted to explore the underlying mechanism of which miR-329 is influencing the progression of CCA. This work studied the differential analysis of the expression chips of CCA obtained from the Gene Expression Omnibus database. Next, to determine both the expression and role of pituitary tumor transforming gene-1 (PTTG1) in CCA, the miRNAs regulating PTTG1 were predicted. In the CCA cells that had been intervened with miR-329 upregulation or inhibition, along with PTTG1 silencing, expression of miR-329, PTTG1, p-p38/p38, p-ERK5/ERK5, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bcl-2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and caspase-3 were determined. The effects of both miR-329 and PTTG1 on cell proliferation, cell-cycle distribution, and apoptosis were also assayed. The miR-329 was likely to affect the CCA development through regulation of the PTTG1-mediated mitogen-activated protein kinase (MAPK) signaling pathway. The miR-329 targeted PTTG1, leading to inactivation of the MAPK signaling pathway. Upregulation of miR-329 and silencing of PTTG1 inhibited the CCA cell proliferation, induced cell-cycle arrest, and subsequently promoted apoptosis with elevations in Bax, cleaved caspase-3, and total caspase-3, but showed declines in PCNA, Cyclin D1, and Bcl-2. Moreover, miR-329 was also found to suppress the tumor growth by downregulation of PTTG1. To summarize, miR-329 inhibited the expression of PTTG1 to inactivate the MAPK signaling pathway, thus suppressing the CCA progression, thereby providing a therapeutic basis for the CCA treatment.

The diagnostic performance of gadoxetic acid disodium-enhanced magnetic resonance imaging and contrast-enhanced multi-detector computed tomography in detecting hepatocellular carcinoma: a meta-analysis of eight prospective studies.

AIM: The purpose of this study was to determine the relative diagnostic benefit of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) over contrast-enhanced multi-detector computed tomography (CEMDCT) for the detection of hepatocellular carcinoma (HCC). METHODS: Two investigators searched multiple databases from inception to January 8, 2019, for studies comparing Gd-EOB-DTPA-enhanced MRI with CEMDCT in adults suspected of HCC. Two reviewers independently selected studies and extracted data. RESULTS: Eight studies were included enrolling 498 patients. MRI showed significantly higher sensitivity than CT (0.85 vs. 0.68). There was no significant difference in the specificity of MRI and CT (0.94 vs. 0.93). The negative likelihood ratio and positive likelihood ratio of MRI and CT were not significantly different (0.16 vs. 0.15 and 14.7 vs. 11.2, respectively). The summary receiver operating characteristics (SROC) of MRI was higher than that of CT at 0.96 vs. 0.91. In the subgroup analysis with a lesion diameter below 2 cm, the sensitivity of MRI was significantly higher than that of CT (0.79 vs. 0.46). CONCLUSION: Gd-EOB-DTPA-enhanced MRI showed higher sensitivity and overall diagnostic accuracy than CEMDCT especially for hepatocellular carcinoma lesions smaller than 2 cm. KEY POINTS: • Gd-EOB-DTPA-enhanced MRI can detect small lesions of hepatocellular carcinoma. • Gd-EOB-DTPA-enhanced MRI showed higher sensitivity and overall diagnostic accuracy than CEMDCT in patients with hepatocellular carcinoma. • Eight prospective studies showed that Gd-EOB-DTPA-enhanced MRI provides greater diagnostic confidence.

IUGR with infantile overnutrition programs an insulin-resistant phenotype through DNA methylation of peroxisome proliferator-activated receptor-γ coactivator-1α in rats.

BACKGROUND: Intrauterine growth restriction (IUGR) followed by postnatal accelerated growth (CG-IUGR) is associated with long-term adverse metabolic consequences, and an involvement of epigenetic dysregulation has been implicated. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a key orchestrator in energy homeostasis. We hypothesized that CG-IUGR programed an insulin-resistant phenotype through the alteration in DNA methylation and transcriptional activity of PGC-1α. METHODS: A CG-IUGR rat model was adopted using maternal gestational nutritional restriction followed by infantile overnutrition achieved by reducing the litter size. The DNA methylation was determined by pyrosequencing. The mRNA expression and mitochondrial content were assessed by real-time PCR. The insulin-signaling protein expression was evaluated by western blotting. RESULTS: Compared with controls, the CG-IUGR rats showed an increase in the DNA methylation of specific CpG sites in PGC-1α, and a decrease in the transcriptional activity of PGC-1α, mitochondrial content, protein level of PI3K and phosphorylated-Akt2 in liver and muscle tissues. The methylation of specific CpG sites in PGC-1α was positively correlated with fasting insulin concentration. CONCLUSION: IUGR followed by infantile overnutrition programs an insulin-resistant phenotype, possibly through the alteration in DNA methylation and transcriptional activity of PGC-1α. The genetic and epigenetic modifications of PGC-1α provide a potential mechanism linking early-life nutrition insult to long-term metabolic disease susceptibilities.

FABP4 deficiency ameliorates alcoholic steatohepatitis in mice via inhibition of p53 signaling pathway.

Fatty acid-binding protein 4 (FABP4) plays an essential role in metabolism and inflammation. However, the role of FABP4 in alcoholic steatohepatitis (ASH) remains unclear. This study aimed to investigate the function and underlying mechanisms of FABP4 in the progression of ASH. We first obtained alcoholic hepatitis (AH) datasets from the National Center for Biotechnology Information-Gene Expression Omnibus database and conducted bioinformatics analysis to identify critical genes in the FABP family. We then established ASH models of the wild-type (WT) and Fabp4-deficient (Fabp4-/-) mice to investigate the role of FABP4 in ASH. Additionally, we performed transcriptional profiling of mouse liver tissue and analyzed the results using integrative bioinformatics. The FABP4-associated signaling pathway was further verified. FABP4 was upregulated in two AH datasets and was thus identified as a critical biomarker for AH. FABP4 expression was higher in the liver tissues of patients with alcoholic liver disease and ASH mice than in the corresponding control samples. Furthermore, the Fabp4-/- ASH mice showed reduced hepatic lipid deposition and inflammation compared with the WT ASH mice. Mechanistically, Fabp4 may be involved in regulating the p53 and sirtuin-1 signaling pathways, subsequently affecting lipid metabolism and macrophage polarization in the liver of ASH mice. Our results demonstrate that Fabp4 is involved in the progression of ASH and that Fabp4 deficiency may ameliorate ASH. Therefore, FABP4 may be a potential therapeutic target for ASH treatment.

Identification of diagnostic markers and molecular clusters of cuproptosis-related genes in alcohol-related liver disease based on machine learning and experimental validation.

Background and aims: Alcohol-related liver disease (ALD) is a worldwide burden. Cuproptosis has been shown to play a key role in the development of several diseases. However, the role and mechanisms of cuproptosis in ALD remain unclear. Methods: The RNA-sequencing data of ALD liver samples were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatical analyses were performed using the R data package. We then identified key genes through multiple machine learning methods. Immunoinfiltration analyses were used to identify different immune cells in ALD patients and controls. The expression levels of key genes were further verified. Results: We identified three key cuproptosis-related genes (CRGs) (DPYD, SLC31A1, and DBT) through an in-depth analysis of two GEO datasets, including 28 ALD samples and eight control samples. The area under the curve (AUC) value of these three genes combined in determining ALD was 1.0. In the external datasets, the three key genes had AUC values as high as 1.0 and 0.917, respectively. Nomogram, decision curve, and calibration curve analyses also confirmed these genes' ability to predict the diagnosis. These three key genes were found to be involved in multiple pathways associated with ALD progression. We confirmed the mRNA expression of these three key genes in mouse ALD liver samples. Regarding immune cell infiltration, the numbers of B cells, CD8 (+) T cells, NK cells, T-helper cells, and Th1 cells were significantly lower in ALD patient samples than in control liver samples. Single sample gene set enrichment analysis (ssGSEA) was then used to estimate the immune microenvironment of different CRG clusters and CRG-related gene clusters. In addition, we calculated CRG scores through principal component analysis (PCA) and selected Sankey plots to represent the correlation between CRG clusters, gene clusters, and CRG scores. Finally, the three key genes were confirmed in mouse ALD liver samples and liver cells treated with ethanol. Conclusions: We first established a prognostic model for ALD based on 3 CRGs and robust prediction efficacy was confirmed. Our investigation contributes to a comprehensive understanding of the role of cuproptosis in ALD, presenting promising avenues for the exploration of therapeutic strategies.

Identification and validation of cuproptosis-related genes in acetaminophen-induced liver injury using bioinformatics analysis and machine learning.

Background: Acetaminophen (APAP) is commonly used as an antipyretic analgesic. However, acetaminophen overdose may contribute to liver injury and even liver failure. Acetaminophen-induced liver injury (AILI) is closely related to mitochondrial oxidative stress and dysfunction, which play critical roles in cuproptosis. Here, we explored the potential role of cuproptosis-related genes (CRGs) in AILI. Methods: The gene expression profiles were obtained from the Gene Expression Omnibus database. The differential expression of CRGs was determined between the AILI and control samples. Protein protein interaction, correlation, and functional enrichment analyses were performed. Machine learning was used to identify hub genes. Immune infiltration was evaluated. The AILI mouse model was established by intraperitoneal injection of APAP solution. Quantitative real-time PCR and western blotting were used to validate hub gene expression in the AILI mouse model. The copper content in the mouse liver samples and AML12 cells were quantified using a colorimetric assay kit. Ammonium tetrathiomolybdate (ATTM), was administered to mouse models and AML12 cells in order to investigate the effects of copper chelator on AILI. Results: The analysis identified 7,809 differentially expressed genes, 4,245 of which were downregulated and 3,564 of which were upregulated. Four optimal feature genes (OFGs; SDHB, PDHA1, NDUFB2, and NDUFB6) were identified through the intersection of two machine learning algorithms. Further nomogram, decision curve, and calibration curve analyses confirmed the diagnostic predictive efficacy of the four OFGs. Enrichment analysis indicated that the OFGs were involved in multiple pathways, such as IL-17 pathway and chemokine signaling pathway, that are related to AILI progression. Immune infiltration analysis revealed that macrophages were more abundant in AILI than in control samples, whereas eosinophils and endothelial cells were less abundant. Subsequently, the AILI mouse model was successfully established, and histopathological analysis using hematoxylin-eosin staining along with liver function tests revealed a significant induction of liver injury in the APAP group. Consistent with expectations, both mRNA and protein levels of the four OFGs exhibited a substantial decrease. The administration of ATTAM effectively mitigates copper elevation induced by APAP in both mouse model and AML12 cells. However, systemic administration of ATTM did not significantly alleviate AILI in the mouse model. Conclusion: This study first revealed the potential role of CRGs in the pathological process of AILI and offered novel insights into its underlying pathogenesis.