Wilson disease (WD) is characterized by hepatic injury caused by copper accumulation; however, the underlying molecular mechanisms remain incompletely understood. This study aims to investigate whether copper overload induces pyroptosis in WD and to elucidate the role of USP9X-mediated JAK1 deubiquitination in this process. Integrated transcriptomic, proteomic, and ubiquitinomic analyses were performed on liver tissues from WD patients and ATP7B-knockout cellular models. The involvement of the JAK1/STAT1 signaling pathway, the CASP9/CASP3/GSDME axis, and USP9X-mediated deubiquitination was further investigated using pharmacological inhibition as well as gene knockout and knockdown approaches, and the findings were subsequently validated in Atp7b−/− mouse models. Copper overload markedly activated the JAK1/STAT1 signaling pathway, enhanced reactive oxygen species (ROS) production, and triggered activation of CASP9 and CASP3, resulting in GSDME cleavage and a switch from hepatocyte apoptosis to pyroptosis. Mechanistically, copper upregulated USP9X expression, which mediated the deubiquitination of JAK1 at lysine 249, thereby stabilizing JAK1 and enhancing downstream signaling cascade. Pharmacological or genetic inhibition of JAK1/STAT1 pathway or GSDME significantly attenuated pyroptosis and alleviated liver injury in WD mouse models, even in the presence of persistent copper accumulation. USP9X deubiquitinates JAK1, thereby promoting copper-induced CASP9/CASP3/GSDME-dependent pyroptosis and liver injury in WD. Targeting the USP9X/JAK1 axis may represent a promising therapeutic strategy for WD. Copper activates the JAK1/STAT1 pathway, leading to ROS production and apoptosis, which in turn induces GSDME-dependent pyroptosis and hepatic injury. Copper also upregulates USP9X expression, thereby stabilizing JAK1 through deubiquitination and further enhancing JAK1/STAT1 signaling and pyroptosis.

Background: Centromere protein F (CENPF), a mitosis-related protein, is overexpressed in hepatocellular carcinoma (HCC) and has emerged as a promising biomarker for early HCC. However, the role of hepatitis B virus (HBV) infection on CENPF overexpression in HCC remains unknown. Moreover, with ultra-large molecular weight of 358kDa of CENPF, no study has directly explored its carcinogenicity with an overexpression model. Materials and Methods: The relationship among HBV infection, CENPF amplification and CENPF overexpression was investigated in HCC tissues. HBV X protein (HBx) transient overexpression cell models was constructed to explore its effect on CENPF expression. CENPF was upregulated and downregulated to analyze its functions in vitro and in vivo. Specifically, a CRISPR/dCas9 system was applied to construct the CENPF overexpression model. Results: A high frequency of CENPF amplification (36.21%, 21/58) was identified in HCC tissues, predominantly in HBVassociated cases (90.48%, 19/21), and CENPF amplification correlated with CENPF overexpression. The HBx enhanced CENPF expression in HBx transfected HCC cells. In addition, CENPF knockdown cell models showed inhibition of HCC proliferation both in vitro and in vivo. Notably, as a cell cycle protein with high constitutive expression in G2/M phase, CENPF overexpression cell models also showed inhibitory effects, probably due to the toxic effect of excessive CENPF expression on G2/M transition. However, in both CENPF downregulation and overexpression models, cell cycle assays showed CENPF promoted G1/S transition in HCC cells. RNA-seq showed that CENPF overexpression activated the MYC pathway, thereby promoting G1/S transition. Rescue experiment indicated that the MYC pathway inhibitor 10058-F4 counteracted the G1/S transition induced by CENPF overexpression in HCC cells. Conclusion: HBV infection was associated with upregulated CENPF expression in HCC and CENPF overexpression might facilitate G1/S transition of HCC cells via the MYC pathway.

BACKGROUND:Obesity impairs liver regeneration by promoting chronic inflammation and metabolic dysfunction, especially in conditions like non-alcoholic fatty liver disease. Portal vein embolization (PVE), used to stimulate liver growth pre-hepatectomy, is less effective in obese subjects. Nicotinamide riboside (NR), a NAD+ precursor, improves mitochondrial function and lipid metabolism, but its role in liver regeneration under obese conditions remains unclear. Our study tried to investigate the effects and underlying mechanisms of NR on liver regeneration after PVE in high-fat diet (HFD)-induced obese rats. METHODS:HFD-fed rats underwent PVE and were treated with or without NR. Liver regeneration was assessed by histology, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, immunohistochemistry, and liver function tests. NAD+ levels were quantified to confirm NR activity. Proteomics, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene Ontology (GO) analysis, quantitative real-time PCR (qPCR), and western blotting were used to explore molecular mechanisms, focusing on the MCART1/ASB3 axis. RESULTS:Obesity impaired liver regeneration post-PVE, as evidenced by lipid accumulation, inflammation, reduced hepatocyte proliferation, and elevated liver enzymes. NR supplementation restored NAD+ levels, improved liver function, increased proliferative activity, and reduced steatosis. Mechanistically, NR upregulated MCART1 and ASB3 expression, promoting energy and lipid metabolism essential for regeneration. CONCLUSIONS:NR promotes liver regeneration after PVE in obese rats by enhancing NAD+-dependent metabolic pathways through the MCART1/ASB3 axis, offering a potential therapeutic strategy for obesity-associated liver dysfunction.

Background:Ribonucleic acid export 1 (RAE1) autoantibody may have good potential for early detection of gastric cancer (GC). However, the carcinogenicity of RAE1 in GC remains unknown. We aimed to explore the role and the potential mechanism for RAE1 in the carcinogenesis of GC. Methods:Immunohistochemical assay was applied to analyze the expression of RAE1 in GC and precancerous lesion (PL) tissues and its relationship with clinical characteristics. The effects of RAE1 on proliferation, migration, apoptosis, and cell cycle were explored by constructing RAE1 knockdown and overexpression in GC cells. The effects of RAE1 knockdown on tumor growth were observed in a murine xenograft model. The signaling pathways involved in GC development that may be affected by RAE1 were investigated by transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results:Immunohistochemical expression of RAE1 was significantly higher in early GC patients than in PL and normal tissues, and the RAE1 expression was correlated with clinical stage (P<0.001). In vitro, knockdown of RAE1 inhibited the proliferation and migration of GC cells with promoted apoptosis and arrested cell cycle in the S phase, while overexpression of RAE1 showed the opposite trend. In vivo, knockdown of RAE1 inhibited the growth of subcutaneous graft tumors in mice. Transcriptome sequencing and subsequent analysis of RAE1 knockdown cells revealed that the Hippo signaling pathway was activated by RAE1 knockdown. Conclusions:RAE1 promotes GC cells proliferation and migration and is associated with the inhibition of the Hippo signaling pathway, and may be a potential biomarker for early diagnosis and treatment of GC.

Background and Aims:Despite advancements in diagnostic and therapeutic strategies, hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality. Antioxidant-1 (ATOX1) has been implicated in oncogenic processes across various cancer types; however, its specific role in HCC remains unclear. This study aimed to investigate the function of ATOX1 and its underlying molecular mechanisms in HCC. Methods:Immunohistochemical analysis was conducted to assess ATOX1 expression in HCC tissues. Cell Counting Kit-8, colony formation, Transwell migration, flow cytometry, and reactive oxygen species (ROS) assays were employed to evaluate the malignant behaviors of tumor cells. A xenograft mouse model was employed to assess the effects of ATOX1 knockdown on tumor growth in vivo. DCAC50 treatment was performed to inhibit the copper transport function of ATOX1. RNA sequencing was conducted to explore the potential molecular mechanisms of ATOX1 in HCC. Results:ATOX1 expression was significantly elevated in HCC tumor tissues. ATOX1 promoted cell proliferation, colony formation, and migration. Knockdown of ATOX1 suppressed tumor growth in vivo. Mechanistically, ATOX1 activated c-Myb, and thus enhanced the malignant phenotype of HCC cells via activation of the PI3K/AKT signaling pathway. Additionally, ATOX1 reduced intracellular copper accumulation and inhibited ROS production and apoptosis. Inhibition of ATOX1 by DCAC50 decreased cell proliferation while increasing ROS levels and apoptosis in HCC cells. Notably, acetylcysteine reversed the reduction in c-Myb expression induced by ATOX1 knockdown. Conclusions:ATOX1 may promote HCC carcinogenesis through the activation of the c-Myb/PI3K/AKT pathway and the inhibition of copper accumulation and oxidative stress.

Resistance to platinum-based chemotherapy agents like oxaliplatin (OXA) poses significant challenges in the treatment of cancers such as hepatocellular carcinoma (HCC). Centrin 2 (CETN2), which functions in nucleotide excision repair (NER) of DNA damage, is overexpressed in HCC. We investigated the potential role of CETN2 in modulating the sensitivity of HCC cells to OXA. CETN2 expression correlated with decreased OXA sensitivity in Huh7 and Hep3B HCC cell lines. CETN2 forms a complex with XPC, which is crucial for the initial DNA damage recognition in NER, thereby enhancing NER and reducing the efficacy of OXA. siRNA-mediated knockdown of CETN2 increased OXA-induced cytotoxicity and apoptosis, confirming its role in chemoresistance. Moreover, overexpression of CETN2 inhibited OXA-induced DNA damage, an effect partially reversed by XPC knockdown. Our findings highlight CETN2 as a potential biomarker and therapeutic target in overcoming OXA resistance in HCC and suggest the possibility for CETN2 inhibitors in enhancing chemotherapeutic efficacy in the treatment of HCC.

BACKGROUND:Hereditary hemochromatosis (HH) is an iron overload disorder and can be caused by variants in non-HFE genes in Chinese patients. However, there is still a considerable proportion of patients suffering from unexplained iron overload. In our previous study, we had identified the p.R269Q variant in exon 4 of the Bone morphogenetic protein 4 (BMP4) gene in Chinese patients with unexplained primary iron overload by Whole Exome sequencing, and then the BMP4 p.H251Y variant was identified by Sanger sequencing in a Chinese patient with secondary iron overload. Our study aimed to explore the pathogenicity and underlying mechanism of BMP4 p.H251Y and BMP4 p.R269Q variants in patients with iron overload. METHODS:Sanger sequencing was conducted to identify the novel variants in the BMP4 gene of patients with unexplained iron overload. MRI and liver biopsy were used to display iron overload in the liver of the patient harboring the BMP4 p.H251Y variant. The BMP4 and hepcidin levels in BMP4 knockdown and BMP4 variant cells were examined by enzyme-linked immunosorbent assay. The effects of BMP4 p.H251Y and BMP4 p.R269Q variants on the hepcidin-regulation pathway were studied. RESULTS:One of 54 HH patients (1.85%) harbored the BMP4 p.R269Q variant. One of 148 patients (0.68%) with secondary hemochromatosis harbored the BMP4 p.H251Y variant, and these two variants were not found in 100 Chinese general population. For the patient harboring the BMP4 p.H251Y variant, abdominal MRI and Perl's staining of liver tissue displayed iron overload in the liver. Cells transfected with the BMP4 p.H251Y and p.R269Q variants showed down-regulation of hepcidin level and BMP/SMAD pathway compared with cells transfected with the wild-type BMP4 vector. CONCLUSION:The BMP4 p.H251Y and p.R269Q variants can downregulate hepcidin levels by inhibiting the BMP/SMAD axis, suggesting they may play pathogenic roles in iron overload.

The spectrum of UDP-glucuronosyltransferase (UGT1A1) variants, which are associated with Gilbert syndrome (GS) and Crigler-Najjar syndrome (CNS-II), has been reported in Chinese and western countries. However, the genotype-phenotype correlation of the individual UGT1A1 variants in GS and CNS-II remains to be clarified. To explore the UGT1A1 variant pattern and genotype-phenotype correlations, we enrolled 310 Chinese patients, including 232 patients with GS and 78 with CNS-II. Peripheral blood samples were collected for screening variants in the gene UGT1A1 by a polymerase chain reaction and Sanger sequencing. The correlation between different UGT1A1 variants and clinical phenotypes was analyzed. A total of 21 UGT1A1 variants were identified, including nine novel variants, and constituted 42 UGT1A1 genotypes in the GS and CNS-II patients. The most common UGT1A1 variants were A(TA)7TAA, p.G71R, p.Y486D, p.P364L, and p.P229Q, which were different from western countries. The p.Y486D variant had higher minor allele frequency in CNS-II than in GS whereas the A(TA)7TAA variant had higher minor allele frequency in GS than in CNS-II. The serum total bilirubin and triglyceride had significant differences among 14 recurrent genotypes of UGT1A1, in which the serum total bilirubin in patients with compound p.Y486D (homozygous)/p.G71R variant was significantly higher compared with homozygous A(TA)7TAA, homozygous p.G71R, compound heterozygous A(TA)7TAA/p.G71R and A(TA)7TAA/p.P364L, and combined heterozygous A(TA)7TAA/p.G71R/p.P229Q, while the serum triglyceride in patients with combined A(TA)7TAA (homozygous)/p.P229Q variant was significantly higher compared with compound heterozygous A(TA)7TAA/p.G71R, single heterozygous A(TA)7TAA, single heterozygous p.G71R, and homozygous A(TA)7TAA. The spectrum of UGT1A1 genotypes in Chinese patients was distinct from western countries. There were differential levels of serum total bilirubin and triglyceride in patients with recurrent genotypes of UGT1A1.

Pathogenic variants in HFE and non- HFE genes have been identified in hemochromatosis in different patient populations, but there are still a certain number of patients with unexplained primary iron overload. We recently identified in Chinese patients a recurrent p.(Arg639Gln) variant in SURP and G-patch domain containing 2 (SUGP2), a potential mRNA splicing-related factor. However, the target gene of SUGP2 and affected iron-regulating pathway remains unknown. We aimed to investigate the pathogenicity and underlying mechanism of this variant in hemochromatosis. RNA-seq analysis revealed that SUGP2 knockdown caused abnormal alternative splicing of CIRBP pre-mRNA, resulting in an increased normal splicing form of CIRBP V1, which in turn increased the expression of BMPER by enhancing its mRNA stability and translation. Furthermore, RNA-protein pull-down and RNA immunoprecipitation assays revealed that SUGP2 inhibited splicing of CIRBP pre-mRNA by a splice site variant at CIRBP c.492 and was more susceptible to CIRBP c.492 C/C genotype. Cells transfected with SUGP2 p.(Arg639Gln) vector showed up-regulation of CIRBP V1 and BMPER expression and down-regulation of pSMAD1/5 and HAMP expression. CRISPR-Cas9 mediated SUGP2 p.(Arg622Gln) knock-in mice showed increased iron accumulation in the liver, higher total serum iron, and decreased serum hepcidin level. A total of 10 of 54 patients with hemochromatosis (18.5%) harbored the SUGP2 p.(Arg639Gln) variant and carried CIRBP c.492 C/C genotype, and had increased BMPER expression in the liver. Altogether, the SUGP2 p.(Arg639Gln) variant down-regulates hepcidin expression through the SUGP2/CIRBP/BMPER axis, which may represent a novel pathogenic factor for hemochromatosis.

Background and Aims:Liver iron overload can induce hepatic expression of bone morphogenic protein (BMP) 6 and activate the BMP/SMAD pathway. However, serum iron overload can also activate SMAD but does not induce BMP6 expression. Therefore, the mechanisms through which serum iron overload activates the BMP/SMAD pathway remain unclear. This study aimed to clarify the role of SMURF1 in serum iron overload and the BMP/SMAD pathway.Methods:A cell model of serum iron overload was established by treating hepatocytes with 2 mg/mL of holo-transferrin (Holo-Tf). A serum iron overload mouse model and a liver iron overload mouse model were established by intraperitoneally injecting 10 mg of Holo-Tf into C57BL/6 mice and administering a high-iron diet for 1 week followed by a low-iron diet for 2 days. Western blotting and real-time PCR were performed to evaluate the activation of the BMP/SMAD pathway and the expression of hepcidin.Results:Holo-Tf augmented the sensitivity and responsiveness of hepatocytes to BMP6. The E3 ubiquitin-protein ligase SMURF1 mediated Holo-Tf-induced SMAD1/5 activation and hepcidin expression; specifically, SMURF1 expression dramatically decreased when the serum iron concentration was increased. Additionally, the expression of SMURF1 substrates, which are important molecules involved in the transduction of BMP/SMAD signaling, was significantly upregulated. Furthermore, in vivo analyses confirmed that SMURF1 specifically regulated the BMP/SMAD pathway during serum iron overload.Conclusions:SMURF1 can specifically regulate the BMP/SMAD pathway by augmenting the responsiveness of hepatocytes to BMPs during serum iron overload.

Purpose Pathogenic variants in HFE and non-HFE genes have been identified in hereditary hemochromatosis (HH) in different patient populations, but there are still a considerable proportion of patients with unexplained primary iron overload. We recently identified in Chinese patients with unexplained primary iron overload a recurrent p.L708V variant in the differentially expressed in normal and neoplastic cells domain 3 (DENND3) gene, functioning as a guanine nucleotide exchange factor for small GTpase Rab12 which down-regulates TfR expression in mice. We aim to investigate the pathogenicity and the underlying mechanism of the DENND3 p.L708V variant in HH patients. Methods Patients with primary iron overload were analyzed for DENND3 p.L708V. TFR2 and hepcidin expression in livers were examined in HH patients harboring DENND3 p.L708V. The effects of DENND3 p.L708V on RAB12/TFR2 and downstream iron metabolic pathways were investigated in vitro and in vivo. Results Six of 31 patients with HH (19.35%) harbored the DENND3 p.L708V variant. The expression of TFR2 and hepcidin was decreased in the liver of HH patients with DENND3 p.L708V. Cells transfected with the DENND3 p.L708V vector showed up-regulation of RAB12 expression and TFR2 degradation in lysosomes, and down-regulation of the pSMAD1/5 and hepcidin. Mice models infected with adeno-associated virus expressing DENND3 p.L708V variant showed higher total serum iron concentrations and decreased HAMP level, increased amount of iron accumulation and the down-regulated of TFR2 expression in the liver. Conclusions The DENND3 p.L708V activating variant down-regulates hepcidin expression through the DENND3/RAB12/TFR2 axis, which may represent a potential novel pathogenic factor of HH.

Supplementary Figure S1, CYP17A1 staining pictures, including 100 pairs of tumor cases in the tissue microarray. Supplementary Figure S2, CYP17A1 staining pictures in 83 informative HCC tissues at grades I to III. Supplementary Figure S3, The SignalP 3.0 server predicted the presence and location of the secretory signal peptide in the amino acid sequence of the human CYP17A1 protein. Supplementary Figure S4, Scheme of the sandwich ELISA and the standard curve for CYP17A1 measurement. Supplementary Figure S5, Receiver operating characteristic (ROC) curves comparing CYP17A1 and AFP in differentiating patients with HCC (n=776) or patients with early HCC (stages I-II, n=305) versus non-HCC subjects (HBV and cirrhosis, n=307).

PURPOSE:Early diagnosis is crucial for optimal prognosis of gastric cancer (GC). Hereby, we aimed to identify novel serum autoantibody-based biomarkers for precancerous lesion (PL) and early GC. METHODS:We performed serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (Nano-LC-Q-TOF-MS/MS) to screen for GC-associated autoantibodies. The identified autoantibodies were analyzed for potential detection value for PL and GC by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves analysis was conducted to evaluate the accuracy of the biomarkers. RESULTS:We identified seven candidates, such as mRNA export factor (RAE1), Nucleophosmin 1 (NPM1), phosphoglycerate kinase 1 (PGK1), and ADP-ribosylation factor 4 (ARF4). Antibodies against all seven proteins were present at higher levels in sera from 242 patients (51 PL, 78 early GC, 113 advanced GC) compared with sera from 122 healthy individuals. RAE1-specific autoantibody discriminated best between patients at different GC stages, with area under the curve (AUC) values of 0.710, 0.745, and 0.804 for PL, early GC, and advanced GC, respectively. Two predictive models composed of gender, RAE1, PGK1, NPM1, and ARF4 autoantibodies (Model 2 for PL) and of age, gender, RAE1, PGK1, and NPM1 autoantibodies (Model 3 for early GC) had improved diagnostic efficiencies, with AUCs of 0.803 and 0.857, sensitivities of 66.7% and 75.6%, and specificities of 78.7% and 87.7%, respectively. CONCLUSION:The identified serum tumor-associated autoantibodies (TAAbs) may have good potential for early detection of GC and PL.

Established taxonomy system based on disease symptom and tissue characteristics have provided an important basis for physicians to correctly identify diseases and treat them successfully. However, these classifications tend to be based on phenotypic observations, lacking a molecular biological foundation. Therefore, there is an urgent to integrate multi-dimensional molecular biological information or multi-omics data to redefine disease classification in order to provide a powerful perspective for understanding the molecular structure of diseases. Therefore, we offer a flexible disease classification that integrates the biological process, gene expression, and symptom phenotype of diseases, and propose a disease-disease association network based on multi-view fusion. We applied the fusion approach to 223 diseases and divided them into 24 disease clusters. The contribution of internal and external edges of disease clusters were analyzed. The results of the fusion model were compared with Medical Subject Headings, a traditional and commonly used disease taxonomy. Then, experimental results of model performance comparison show that our approach performs better than other integration methods. As it was observed, the obtained clusters provided more interesting and novel disease-disease associations. This multi-view human disease association network describes relationships between diseases based on multiple molecular levels, thus breaking through the limitation of the disease classification system based on tissues and organs. This approach which motivates clinicians and researchers to reposition the understanding of diseases and explore diagnosis and therapy strategies, extends the existing disease taxonomy. Availability of data and materials: The preprocessed dataset and source code supporting the conclusions of this article are available at GitHub repository https://github.com/yangxiaoxi89/mvHDN.