Dysfunction of ATP7B Splicing Variant Caused by Enhanced Interaction With COMMD1 in Wilson Disease.

BACKGROUND & AIMS: The association between Wilson disease and various ATP7B mutations is well-established; however, the molecular mechanism underlying the functional consequence of these mutations, particularly the splicing mutations, remains unclear. This study focused on the ATP7B c.1543+1G>C variant, to reveal a universal pathogenic mechanism of the ATP7B mutants with altered N-terminus. METHODS: The splicing assay and RNA pull-down were performed to explore the mechanism of the aberrant splicing. The ATP7B knockout HuH-7 cell line and Atp7b-/- mice were created, and the functional consequence of the mutant ATP7B were evaluated in vitro and in vivo. RESULTS: The c.1543+1G>C mutation resulted in the skipping of ATP7B exon 3, and the mutant ATP7B showed a loss of trans-Golgi network localization and was degraded via the ubiquitin-proteasome pathway, facilitated by enhanced interactions with COMMD1. Elevated intercellular copper concentration and reduced survival rate were observed in HuH-7 cells expressing mutant ATP7B. Restoration of wild-type ATP7B in Atp7b-/- mice resulted in a substantial improvement in phenotype, whereas mice treated with mutant ATP7B did not demonstrate equivalent benefits. CONCLUSIONS: Our research investigated the pathogenicity and mechanism of ATP7B c.1543+1G>C variant, with particular focus on its enhanced interaction with COMMD1 as a potential universal mechanism contributing to the dysfunction of various ATP7B variants. These findings provide a foundation for the development of innovative therapeutic strategies that target abnormal splicing events in a range of hereditary diseases, including Wilson disease.

Analysis of UGT1A1 genotype-phenotype correlation in Chinese patients with gilbert and crigler-Najjar II syndrome.

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.

SUGP2 p.(Arg639Gln) variant is involved in the pathogenesis of hemochromatosis via the CIRBP/BMPER signaling pathway.

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.

Serum Iron Overload Activates the SMAD Pathway and Hepcidin Expression of Hepatocytes via SMURF1.

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.

Flagellar hook protein FlgE promotes macrophage activation and atherosclerosis by targeting ATP5B.

BACKGROUND AND AIMS: Pseudomonas aeruginosa (P. aeruginosa) infections are strongly linked to the development of cardiovascular disease and atherosclerosis; however, the underlying mechanisms remain unclear. We previously confirmed that the flagellar hook protein FlgE in P. aeruginosa has immunostimulatory effects. This study investigated the effects and mechanisms of action of FlgE on atherogenesis. METHODS: ApoE-/- mice were intravenously challenged with FlgE or FlgEM recombinant proteins for eight weeks. A murine model of chronic lung colonization was established using beads containing either mutable- or wild-type bacteria. Aortic sinus sections were stained to assess atherosclerosis progression. THP-1 macrophages exposed to FlgE or FlgEM were evaluated for their effects on lipid uptake and inflammation in vitro. Western blotting and pull-down assays were used to identify the binding proteins and signaling pathways involved, and specific blocking experiments were performed to confirm these effects. RESULTS: FlgE accelerated atherosclerosis progression by triggering lipid deposition and inflammatory responses in high-fat diet (HFD)-fed ApoE-/- mice. In comparison to infection with wild-type PAO1, infection with PAO1/flgEΔBmF resulted in reduced atherosclerosis. Mechanistic analysis indicated that FlgE exacerbated lipoprotein uptake and foam cell formation by upregulating SR-A1 expression. Moreover, FlgE activated NF-κB and MAPK signaling, which subsequently led to inflammatory responses in THP-1-derived macrophages. Pull-down assays revealed that FlgE directly interacted with ATP5B, whereas blocking ATP5B attenuated FlgE-induced responses in macrophages. CONCLUSIONS: FlgE induces macrophage lipid uptake and pro-inflammatory responses mediated by ATP5B/NF-kB/AP-1 signaling, which eventually results in atherosclerosis. These findings support the development of therapeutic strategies for P. aeruginosa infection-induced atherosclerosis.

Identification of novel serum autoantibody biomarkers for early esophageal squamous cell carcinoma and high-grade intraepithelial neoplasia detection.

Background: Early diagnosis of esophageal squamous cell carcinoma (ESCC) is critical for effective treatment and optimal prognosis; however, less study on serum biomarkers for the early ESCC detection has been reported. The aim of this study was to identify and evaluate several serum autoantibody biomarkers in early ESCC. Methods: We initially screened candidate tumor-associated autoantibodies (TAAbs) associated with ESCC by serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (nano-LC-Q-TOF-MS/MS), and the TAAbs were further subjected to analysis by Enzyme-linked immunosorbent assay (ELISA) in a clinical cohort (386 participants, including 161 patients with ESCC, 49 patients with high-grade intraepithelial neoplasia [HGIN] and 176 healthy controls [HC]). Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance. Results: The serum levels of CETN2 and POFUT1 autoantibodies which were identified by SERPA were statistically different between ESCC or HGIN patients and HC in ELISA analysis with the area under the curve (AUC) values of 0.709 (95%CI: 0.654-0.764) and 0.741 (95%CI: 0.689-0.793), 0.717 (95%CI: 0.634-0.800) and 0.703 (95%CI: 0.627-0.779) for detection of ESCC and HGIN, respectively. Combining these two markers, the AUCs were 0.781 (95%CI: 0.733-0.829), 0.754 (95%CI: 0.694-0.814) and 0.756 (95%CI: 0.686-0.827) when distinguishing ESCC, early ESCC and HGIN from HC, respectively. Meanwhile, the expression of CETN2 and POFUT1 was found to be correlated with ESCC progression. Conclusions: Our data suggest that CETN2 and POFUT1 autoantibodies have potential diagnostic value for ESCC and HGIN, which may provide novel insights for early ESCC and precancerous lesions detection.

Identification and evaluation of novel serum autoantibody biomarkers for early diagnosis of gastric cancer and precancerous lesion.

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.

MARVELD3 inhibits the epithelial-mesenchymal transition and cell migration by suppressing the Wnt/ß-catenin signaling pathway in non-small cell lung cancer cells.

BACKGROUND: The epithelial-mesenchymal transition (EMT), featured by abatement of cell-cell contact, is related to exacerbating non-small cell lung cancer (NSCLC) by inducing metastasis. MAL and relevant proteins for vesicle trafficking and membrane link domain 3 (MARVELD3) is a novel tight junction protein participated in the EMT. There is limited information available about the mechanism of MARVELD3 in NSCLC. In this trial, the inhibition effect of MARVELD3 on human NSCLC cells will be discussed. METHODS: MARVELD3 expression was measured in NSCLC tissues and para-carcinoma tissues. The expression of MARVELD3 and EMT-related genes were examined in transforming growth factor (TGF)-ß1-induced NSCLC cells. NSCLC cells with MARVELD3-knockdown and overexpressed were established to analyze the relationship between MARVELD3 and EMT and cell migration. The Wnt/ß-catenin pathway expression was also analyzed in NSCLC cell models and clinic species. RESULTS: Lower protein levels of MARVELD3 were observed in NSCLC samples than para-carcinoma specimens, and the decreased expression of MARVELD3 in NSCLC specimens was associated with tumor metastasis. E-Cadherin and MARVELD3 expression was reduced in TGF-ß1 treated NSCLC cells, whereas increased Vimentin expression was detected. MARVELD3 changed the EMT-related genes and induced cell migration. In addition, Wnt/ß-catenin pathway and target genes, MYC and CCND1, expressions were inhibited in MARVELD3 overexpressed NSCLC cells. CONCLUSIONS: TGF-ß1 induced EMT in human NSCLC cells can be suppressed by MARVELD3 through Wnt/ß-catenin signaling pathway. These results indicate that MARVELD3 might be a potential therapeutic modality useful in the treatment of NSCLC.

DENND3 p.L708V activating variant is involved in the pathogenesis of hereditary hemochromatosis via the RAB12/TFR2 signaling pathway.

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.

An iron-dependent form of non-canonical ferroptosis induced by labile iron.

Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death characterized by reactive oxygen species (ROS) generation and lipid peroxidation. Here, we report a novel iron-dependent form of ferroptosis induced by labile iron and investigate the mechanism underlying this process. We find that labile iron-induced ferroptosis is distinct from canonical ferroptosis and is linked to the mitochondrial pathway. Specifically, the mitochondrial calcium uniporter mediates the ferroptosis induced by labile iron. Interestingly, cells undergoing labile iron-induced ferroptosis exhibit cytoplasmic features of oncosis and nuclear features of apoptosis. Furthermore, labile iron-induced ferroptosis involves a unique set of genes. Finally, labile iron-induced ferroptosis was observed in liver subjected to acute iron overload in vivo. Our study reveals a novel form of ferroptosis that may be implicated in diseases caused by acute injury.

Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

BACKGROUND AND AIMS: Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. METHODS: An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. RESULTS: Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. CONCLUSIONS: Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Given that HCC is an extraordinarily heterogeneous malignant disease, finding an effective therapeutic strategy for treating it has been difficult. Because of the importance of angiogenesis in tumorigenesis, targeting the more homogenous HCC endothelial cells may be a better therapeutic strategy. In a unpublished manuscript, we found that the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase 2/9 (MMP2/9) were reduced in human HCC tissues that overexpressed DNA damage repair gene general transcription factor II subunit H2 (GTF2H2). This suggested that GTF2H2 may have an inhibitory effect on angiogenesis. Therefore, we hypothesized that GTF2H2 acts as an anti-angiogenesis gene. However, our results showed that GTF2H2 overexpression had no effect on endothelial cell viability, migration, or permeability. To our surprise, treating human umbilical vein endothelial cells (HUVECs) with the culture medium of Huh 7 cells overexpressing GTF2H2 could inhibit their viability, migration, and permeability. We then isolated the culture medium into exosomes and other components from the culture medium. Only GTF2H2-enriched exosomes could inhibit the viability, migration, tube formation, and permeability of HUVECs. Our results suggest that overexpressing GTF2H2 had no effect on HUVECs, while GTF2H2 enriched exosomes from Huh7 cells could inhibit HUVEC phenotypes such as proliferation and migration. Therefore, GTF2H2-enriched exosomes can possibly be utilized as a novel drug for treating HCC and also serve as a potential molecular target for inhibiting tumor angiogenesis.

Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis.

BACKGROUNDS: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. METHODS: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. RESULTS: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. CONCLUSIONS: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Identification of potential modifier genes in Chinese patients with Wilson disease.

The mutations in modifier genes may contribute to some inherited diseases including Wilson disease (WD). This study was designed to identify potential modifier genes that contribute to WD. A total of 10 WD patients with single or no heterozygous ATP7B mutations were recruited for whole-exome sequencing (WES). Five hundred and thirteen candidate genes, of which the genetic variants present in at least two patients, were identified. In order to clarify which proteins might be involved in copper transfer or metabolism processes, the isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify the differentially expressed proteins between normal and CuSO4-treated cell lines. Thirteen genes/proteins were identified by both WES and iTRAQ, indicating that disease-causing variants of these genes may actually contribute to the aberrant copper ion accumulation. Additionally, the c.86C > T (p.S29L) mutation in the SLC31A2 gene (coding CTR2) has a relative higher frequency in our cohort of WD patients (6/191) than reported (0.0024 in gnomAD database) in our healthy donors (0/109), and CTR2S29L leads to increased intracellular Cu concentration and Cu-induced apoptosis in cultured cell lines. In conclusion, the WES and iTRAQ approaches successfully identified several disease-causing variants in potential modifier genes that may be involved in the WD phenotype.

Disparate genomic characteristics of patients with early-stage lung adenocarcinoma manifesting as radiological subsolid or solid lesions.

INTRODUCTION: Early-stage lung adenocarcinoma (LUAD) manifesting as subsolid nodules (SSNs) exhibit more favorable prognosis than solid nodules (SNs). However, the genomic underpinnings behind their indolent tumor behavior remain largely unexplained. METHODS: We identified patients with stage I invasive LUAD who underwent complete surgical resection and broad-panel next-generation sequencing (NGS). Comparative genomic profiling was then performed by radiological subtype (SSNs vs. SNs) regarding the general genomic features, driver genes, oncogenic pathways, therapeutic actionability, and evolutionary trajectory. RESULTS: In total, 177 SSN-LUADs and 133 SN-LUADs were included. Compared with SNs, SSN-LUADs possessed lower somatic mutation count (P < 0.001), genomic alteration count (P = 0.002), and intra-tumor heterogeneity (P = 0.006). In terms of driver genes, SSNs harbored more EGFR mutation (77% vs. 62%), but had lower frequencies of genes such as TP53, ARID1A, PIK3CA, CDKN2A, and BRAF (FDR q < 0.1). Besides, RBM10 mutation was independently associated with SSN-LUADs in multivariate analysis (P = 0.033). Three oncogenic pathways (p53, cell cycle, PI3K) were altered with statistical significance in SNs, while only RNA splicing/processing pathway was significantly altered in SSNs (FDR q < 0.1). Also, SSNs had significantly lower number of pathway alterations (P < 0.001). Finally, SSNs and SNs showed distinct evolutionary trajectories regarding somatic mutations during early-stage LUAD progression. CONCLUSIONS: This study performed the first direct comparative genomic profiling in pathologic stage I invasive LUAD by radiological subtype, highlighting a less complex genomic architecture of SSNs, which might be the molecular interpretation of their indolent tumor behavior.

Single-cell RNA sequencing reveals the multi-cellular ecosystem in different radiological components of pulmonary part-solid nodules.

BACKGROUND: Early-stage lung adenocarcinoma that radiologically manifests as part-solid nodules, consisting of both ground-glass and solid components, has distinctive growth patterns and prognosis. The characteristics of the tumour microenvironment and transcriptional features of the malignant cells of different radiological phenotypes remain poorly understood. METHODS: Twelve treatment-naive patients with radiological part-solid nodules were enrolled. After frozen pathology was confirmed as lung adenocarcinoma, two regions (ground-glass and solid) from each of the 12 part-solid nodules and 5 normal lung tissues from 5 of the12 patients were subjected to single-cell sequencing by 10x Genomics. We used Seurat v3.1.5 for data integration and analysis. RESULTS: We comprehensively dissected the multicellular ecosystem of the ground-glass and solid components of part-solid nodules at the single-cell resolution. In tumours, these components had comparable proportions of malignant cells. However, the angiogenesis, epithelial-to-mesenchymal transition, KRAS, p53, and cell-cycle signalling pathways were significantly up-regulated in malignant cells within solid components compared to those within ground-glass components. For the tumour microenvironment, the relative abundance of myeloid and NK cells tended to be higher in solid components than in ground-glass components. Slight subtype composition differences existed between the ground-glass and solid components. The T/NK cell subsets' cytotoxic function and the macrophages' pro-inflammation function were suppressed in solid components. Moreover, pericytes in solid components had a stronger communication related to angiogenesis promotion with endothelial cells and tumour cells. CONCLUSION: The cellular landscape of ground-glass components is significantly different from that of normal tissue and similar to that of solid components. However, transcriptional differences exist in the vital signalling pathways of malignant and immune cells within these components.

Cancer biology deciphered by single-cell transcriptomic sequencing.

Tumors are complex ecosystems in which heterogeneous cancer cells interact with their microenvironment composed of diverse immune, endothelial, and stromal cells. Cancer biology had been studied using bulk genomic and gene expression profiling, which however mask the cellular diversity and average the variability among individual molecular programs. Recent advances in single-cell transcriptomic sequencing have enabled a detailed dissection of tumor ecosystems and promoted our understanding of tumorigenesis at single-cell resolution. In the present review, we discuss the main topics of recent cancer studies that have implemented single-cell RNA sequencing (scRNA-seq). To study cancer cells, scRNA-seq has provided novel insights into the cancer stem-cell model, treatment resistance, and cancer metastasis. To study the tumor microenvironment, scRNA-seq has portrayed the diverse cell types and complex cellular states of both immune and non-immune cells interacting with cancer cells, with the promise to discover novel targets for future immunotherapy.

Apoptosis-induced translocation of centromere protein F in its corresponding autoantibody production in hepatocellular carcinoma.

Serum autoantibodies against tumor-associated antigen have important value in the early diagnosis of hepatocellular carcinoma (HCC), but the mechanism of autoantibody production is poorly understood. We previously showed that autoantibodies against the centromere protein F (CENPF) may be useful as an early diagnostic marker for HCC. Here we explored the mechanism of cell apoptosis-based CENPF autoantibody production and verified the correlation of CENPF autoantibody level with HCC development. We demonstrated that CENPF was overexpressed and aberrantly localized throughout the nuclei and cytoplasm in human HCC cells compared with hepatic cells. CENPF overexpression promoted the production of CENPF autoantibodies in a manner that correlated with tumor growth of mouse HCC model. During apoptosis of HCC cells, CENPF protein translocated to apoptotic vesicles and relocalized at the cell surface. Through isolating apoptotic components, we found apoptotic body and blebs with lower CD31 and CD47 expression more effectively induced DC phagocytosis and maturation compared with apoptotic intact cells in vitro, and this DC response was independent of CENPF expression. Moreover, injection of mice with apoptotic bodies and blebs effectively induced an immune response and the production of CENPF-specific antibodies. Our findings provide a first elucidation of mechanisms underlying the CENPF autoantibody production via cell apoptosis-induced CENPF translocation, and demonstrate a direct correlation between CENPF autoantibody levels and HCC progression, suggesting the potential of CENPF autoantibody as an HCC diagnostic marker.

Correlation of genotype and phenotype in 32 patients with hereditary hemochromatosis in China.

BACKGROUND: Hereditary hemochromatosis (HH) is widely recognized and clinical manifestations of hemochromatosis-related (HFE-related) HH is well studied in European populations. Less is known about the clinical and laboratory characteristics of non-HFE related HH in Asian population. We aimed to explore the relationship between genotype and clinical phenotype in Chinese patients with non-HFE related hereditary hemochromatosis. METHODS: Peripheral blood samples and clinical data of patients with primary iron overload were collected from the China Registry of Genetic/Metabolic Liver Diseases. Sanger sequencing was performed in cases with primary iron overload, for 5 known HH related genes (HFE, HJV, HAMP, TFR2 and SLC40A1) and 2 novel iron homeostasis-related genes (DENND3 and SUGP2). The correlation of genotype and clinical phenotype in these patients was analyzed. RESULTS: Of the 32 patients with primary iron overload (23 were males and 9 were females), non-HFE variants were detected in 31 (31/32, 97%), including 8 pathogenic variants in HJV, 7 pathogenic variants in SLC40A1, 8 likely pathogenic variants in SUGP2 and 5 likely pathogenic variants in DENND3 cases. Among these 31 cases, 4 cases harbored homozygous variants, 2 cases harbored homozygous + heterozygous variants, 19 cases harbored heterozygous or combined heterozygous variants, and 6 cases harbored no any damaging variants. None of investigated cases carried damaging HAMP and TFR2 variants were found. 8 cases were classified as type 2A HH and 6 cases as type 4 HH, 10 cases as non-classical genotype, and 6 cases had no pathogenic variants from 31 cases. During the statistical analysis, we excluded one case (SLC40A1 IVS3 + 10delGTT + SUGP2 p. R639Q(homo)) with difficulty in grouping due to combined damaging variants. Cases with type 2A HH have an earlier age at diagnosis (p = 0.007). The iron index of cases in type 2A HH and type 4 HH was higher than that in other groups (p = 0.01). Arthropathy was relatively rare in all groups. None of cases with type 2A HH developed cirrhosis. Cirrhosis and diabetes are more prevalent in type 4 HH. The incidence of cirrhosis (p = 0.011), cardiac involvement (p = 0.042), diabetes (p = 0.035) and hypogonadism (p = 0.020) was statistically significant in the four groups. However, due to the limited sample size, the pairwise comparison showed no significant difference. CONCLUSIONS: This is the first comprehensive analysis about the gene variant spectrum and phenotypic aspects of non-HFE HH in China. The results will be useful to the identification, diagnosis and management of HH in China.

Role of tight junction-associated MARVEL protein marvelD3 in migration and epithelial-mesenchymal transition of hepatocellular carcinoma.

MarvelD3, a recently identified tight junction membrane protein, could be associated with hepatocellular carcinoma (HCC). We aimed to investigate the role of marvelD3 in Epithelial-Mesenchymal Transition (EMT) and migration of HCC and explore the underlying molecular mechanisms. First, we assessed marvlD3 expression in HCC and normal liver tissues and found loss of marvelD3 expression was significantly correlated with the occurrence and TNM stage of HCC. Second, we detected that marvelD3 was downregulated in HCC cells with transforming growth factor ß1 and snail/slug-induced EMT. Finally, we analyzed expression of marvelD3 protein was significantly associated with EMT and the NF-κB signaling pathway. Our study demonstrated that MarvelD3 inhibited EMT and migration of HCC cells along with inhibiting NF-κB signaling pathway.Abbreviations:HCC, Hepatocellular carcinoma; TJ, Tight junction; MARVEL, MAL and related proteins for vesicle trafficking and membrane link; EMT, Epithelial-mesenchymal transition; NF-κB, Nuclear factor kappa B; TAMPs, Tight junction-associated marvel proteins; TGF-ß1, Transforming growth factor-ß1; MMP9, matrix metallopeptidase 9; RT-PCR, Real-time PCR; IHC, Immunohistochemistry; IF, Immunofluorescence.