Identification and validation of a novel predictive signature based on hepatocyte-specific genes in hepatocellular carcinoma by integrated analysis of single-cell and bulk RNA sequencing.

BACKGROUND: Hepatocellular carcinoma represents a significant global burden in terms of cancer-related mortality, posing a substantial risk to human health. Despite the availability of various treatment modalities, the overall survival rates for patients with hepatocellular carcinoma remain suboptimal. The objective of this study was to explore the potential of novel biomarkers and to establish a novel predictive signature utilizing multiple transcriptome profiles. METHODS: The GSE115469 and CNP0000650 cohorts were utilized for single cell analysis and gene identification. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets were utilized in the development and evaluation of a predictive signature. The expressions of hepatocyte-specific genes were further validated using the GSE135631 cohort. Furthermore, immune infiltration results, immunotherapy response prediction, somatic mutation frequency, tumor mutation burden, and anticancer drug sensitivity were analyzed based on various risk scores. Subsequently, functional enrichment analysis was performed on the differential genes identified in the risk model. Moreover, we investigated the expression of particular genes in chronic liver diseases utilizing datasets GSE135251 and GSE142530. RESULTS: Our findings revealed hepatocyte-specific genes (ADH4, LCAT) with notable alterations during cell maturation and differentiation, leading to the development of a novel predictive signature. The analysis demonstrated the efficacy of the model in predicting outcomes, as evidenced by higher risk scores and poorer prognoses in the high-risk group. Additionally, a nomogram was devised to forecast the survival rates of patients at 1, 3, and 5 years. Our study demonstrated that the predictive model may play a role in modulating the immune microenvironment and impacting the anti-tumor immune response in hepatocellular carcinoma. The high-risk group exhibited a higher frequency of mutations and was more likely to benefit from immunotherapy as a treatment option. Additionally, we confirmed that the downregulation of hepatocyte-specific genes may indicate the progression of hepatocellular carcinoma and aid in the early diagnosis of the disease. CONCLUSION: Our research findings indicate that ADH4 and LCAT are genes that undergo significant changes during the differentiation of hepatocytes into cancer cells. Additionally, we have created a unique predictive signature based on genes specific to hepatocytes.

The micro-743a-3p-GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice.

BACKGROUND AND AIMS: Epidemiological evidence suggests that the phenotype of glutathione S-transferase mu 1 (GSTM1), a hepatic high-expressed phase II detoxification enzyme, is closely associated with the incidence of alcohol-related liver disease (ALD). However, whether and how hepatic GSTM1 determines the development of ALD is largely unclear. This study was designed to elucidate the role and potential mechanism(s) of hepatic GSTM1 in the pathological process of ALD. METHODS: GSTM1 was detected in the liver of various ALD mice models and cultured hepatocytes. Liver-specific GSTM1 or/and micro (miR)-743a-3p deficiency mice were generated by adenoassociated virus-8 delivered shRNA, respectively. The potential signal pathways involving in alcohol-regulated GSTM1 and GSTM1-associated ALD were explored via both genetic manipulation and pharmacological approaches. RESULTS: GSTM1 was significantly upregulated in both chronic alcohol-induced mice liver and ethanol-exposed murine primary hepatocytes. Alcohol-reduced miR-743a-3p directly contributed to the upregulation of GSTM1, since liver specific silencing miR-743a-3p enhanced GSTM1 and miR-743a-3p loss protected alcohol-induced liver dysfunctions, which was significantly blocked by GSTM1 knockdown. GSTM1 loss robustly aggravated alcohol-induced hepatic steatosis, oxidative stress, inflammation, and early fibrotic-like changes, which was associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38. GSTM1 antagonized ASK1 phosphorylation and its downstream JNK/p38 signaling pathway upon chronic alcohol consumption via binding with ASK1. ASK1 blockage significantly rescued hepatic GSTM1 loss-enhanced disorders in alcohol-fed mice liver. CONCLUSIONS: Chronic alcohol consumption-induced upregulation of GSTM1 in the liver provides a feedback protection against hepatic steatosis and liver injury by counteracting ASK1 activation. Down-regulation of miR-743a-3p improves alcohol intake-induced hepatic steatosis and liver injury via direct targeting on GSTM1. The miR-743a-3p-GSTM1 axis functions as an innate protective pathway to defend the early stage of ALD.

Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Immunomodulation in non-alcoholic fatty liver disease: exploring mechanisms and applications.

Non-alcoholic fatty liver disease (NAFLD) exhibits increased lipid enrichment in hepatocytes. The spectrum of this disease includes stages such as nonalcoholic simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), and liver fibrosis. Changes in lifestyle behaviors have been a major factor contributing to the increased cases of NAFLD patients globally. Therefore, it is imperative to explore the pathogenesis of NAFLD, identify therapeutic targets, and develop new strategies to improve the clinical management of the disease. Immunoregulation is a strategy through which the organism recognizes and eliminates antigenic foreign bodies to maintain physiological homeostasis. In this process, multiple factors, including immune cells, signaling molecules, and cytokines, play a role in governing the evolution of NAFLD. This review seeks to encapsulate the advancements in research regarding immune regulation in NAFLD, spanning from underlying mechanisms to practical applications.

Dynamics of cellular plasticity in non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is a pathogenic stage of the broader non-alcoholic fatty liver disease (NAFLD). Histological presentation of NASH includes hepatocyte ballooning, macrophage polarization, ductular reaction, and hepatic stellate cell (HSCs) activation. At a cellular level, a heterogenous population of cells such as hepatocytes, macrophages, cholangiocytes, and HSCs undergo dramatic intra-cellular changes in response to extracellular triggers, which are termed "cellular plasticity. This dynamic switch in the cellular structure and function of hepatic parenchymal and non-parenchymal cells and their crosstalk culminates in the perpetuation of inflammation and fibrosis in NASH. This review presents an overview of our current understanding of cellular plasticity in NASH and its molecular mechanisms, along with possible targeting to develop cell-specific NASH therapies.

Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice.

Diethylnitrosamine (DEN) is a chemical hepatocarcinogenic agent that triggers a large array of oncogenic mutations after a single injection. Initiated hepatocytes subsequently undergo clonal expansion within a proliferative environment, rendering the DEN model a comprehensive carcinogen. In rodent studies, DEN finds extensive utility in experimental liver cancer research, mimicking several aspects of human hepatocellular carcinoma (HCC), including angiogenesis, metabolic reprogramming, immune exhaustion, and the ability to metastasize. Beyond the wealth of scientific insights gleaned from this model, the objective of this chapter is to review morphological, genomic, and immunological characteristics associated to DEN-induced HCC. Furthermore, this chapter provides a detailed procedural guide to effectively induce hepatocarcinogenesis in mice through a single intraperitoneal injection of DEN.

The relationship and diagnostic efficacy of N-terminal propeptide type III collagen in pathological changes associated with non-alcoholic steatohepatitis.

OBJECTIVE: This study delves into the role of N-terminal propeptide type III collagen (PIIINP) in the diagnosis and management of liver pathological changes associated with non-alcoholic steatohepatitis (NASH). PATIENTS AND METHODS: We collected baseline information, pathological data, and serum PIIINP levels of 168 patients diagnosed with non-alcoholic fatty liver disease (NAFLD) via ultrasound imaging in our hospital. Based on the non-alcoholic fatty liver disease activity score (NAS), patients with different NAFLD patterns were divided into a Definite NASH group and a Not/borderline group. Differences in PIIINP levels and pathological features between the two groups were compared and analyzed. The diagnostic value of PIIINP for NASH was evaluated using the receiver operating characteristic (ROC) curve. RESULTS: Patients with NASH exhibited significantly higher values of homeostatic model assessment for insulin resistance (HOMA-IR), fibrosis biomarker fibrosis-4 (FIB-4), aminotransferase-to-platelet ratio index (APRI), and serum PIIINP levels than those classified as Not/borderline. A marked increase in the serum concentrations of PIIINP was observed with the severity of fatty degeneration, lobular inflammation, and hepatocellular ballooning. The AUC of PIIINP for diagnosing definite NASH was 0.766 (95% CI: 0.694, 0.839), APRI was 0.634 (95% CI: 0.549, 0.718), and FIB-4 was 0.621 (95% CI: 0.534, 0.708). The AUC of PIIINP for diagnosing definite NASH was significantly higher than that of APRI and FIB-4 (all p<0.05). Utilizing the predetermined threshold values for diagnostic parameters, the PIIINP measure demonstrated a sensitivity of 71.6% and a specificity of 73.6% in diagnosing definitive NASH when its value exceeded 7.72 ng/dL. This yielded a Youden index of 0.45. Similarly, when the APRI measure exceeded 0.21, it exhibited a sensitivity of 60.5% and a specificity of 63.2%, resulting in a Youden index of 0.24. Moreover, when the FIB-4 index surpassed 0.26, it showed a sensitivity of 46.9% and a specificity of 79.3%, culminating in a Youden index of 0.26. CONCLUSIONS: NASH patients in this study exhibited significantly elevated PIIINP serum levels, which were closely associated with hepatocyte pathological changes. PIIINP demonstrated superior competence in diagnosing NASH than APRI and FIB-4 and thus offers a viable alternative for the clinical diagnosis of NASH.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. METHODS: In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. RESULTS: We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. CONCLUSION: We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.

Liver fibrosis: pathological features, clinical treatment and application of therapeutic nanoagents.

Liver fibrosis is a reversible damage-repair response, the pathological features of which mainly include damage to hepatocytes, sinusoid capillarization, hepatic stellate cells activation, excessive accumulation of extracellular matrix and inflammatory response. Although some treatments (including drugs and stem cell therapy) for these pathological features have been shown to be effective, more clinical trials are needed to confirm their effectiveness. In recent years, nanomaterials-based therapies have emerged as an innovative and promising alternative to traditional drugs, being explored for the treatment of liver fibrosis diseases. Natural nanomaterials (including extracellular vesicles) and synthetic nanomaterials (including inorganic nanomaterials and organic nanomaterials) are developed to facilitate drug targeting delivery and combination therapy. In this review, the pathological features of liver fibrosis and the current anti-fibrosis drugs in clinical trials are briefly introduced, followed by a detailed introduction of the therapeutic nanoagents for the precise delivery of anti-fibrosis drugs. Finally, the future development trend in this field is discussed.

(+)-Lipoic acid reduces mitochondrial unfolded protein response and attenuates oxidative stress and aging in an in vitro model of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a liver disorder characterized by the ac-cumulation of fat in hepatocytes without alcohol consumption. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play significant roles in NAFLD pathogenesis. The unfolded protein response in mitochondria (UPRmt) is an adaptive mechanism that aims to restore mitochondrial protein homeostasis and mitigate cellular stress. This study aimed to investigate the effects of ( +)-Lipoic acid (ALA) on UPRmt, inflammation, and oxidative stress in an in vitro model of NAFLD using HepG2 cells treated with palmitic acid and oleic acid to induce steatosis. RESULTS: Treatment with palmitic and oleic acids increased UPRmt-related proteins HSP90 and HSP60 (heat shock protein), and decreased CLPP (caseinolytic protease P), indicating ER stress activation. ALA treatment at 1 µM and 5 µM restored UPRmt-related protein levels. PA:OA (palmitic acid:oleic acid)-induced ER stress markers IRE1α (Inositol requiring enzyme-1), CHOP (C/EBP Homologous Protein), BIP (Binding Immunoglobulin Protein), and BAX (Bcl-2-associated X protein) were significantly reduced by ALA treatment. ALA also enhanced ER-mediated protein glycosylation and reduced oxidative stress, as evidenced by decreased GPX1 (Glutathione peroxidase 1), GSTP1 (glutathione S-transferase pi 1), and GSR (glutathione-disulfide reductase) expression and increased GSH (Glutathione) levels, and improved cellular senescence as shown by the markers ß-galactosidase, γH2Ax and Klotho-beta. CONCLUSIONS: In conclusion, ALA ameliorated ER stress, oxidative stress, and inflammation in HepG2 cells treated with palmitic and oleic acids, potentially offering therapeutic benefits for NAFLD providing a possible biochemical mechanism underlying ALA beneficial effects.

miR-155-5p promotes hepatic steatosis via PICALM-mediated autophagy in aging hepatocytes.

BACKGROUND: Hepatic steatosis, a lipid disorder characterized by the accumulation of intrahepatic fat, is more prevalent in the elderly population. This study investigates the role of miR-155-5p in the autophagy dysregulation of aging hepatic steatosis. METHODS: We established an aging mouse model in vivo and a hepatocellular senescence model induced by low serum and palmitic acid in vitro. The fluctuations of microRNAs were derived from RNA-seq data and confirmed by qPCR in 4- and 18-month-old mouse liver tissues. Hematoxylin-eosin (H&E) staining observed pathological changes. Markers of senescence, autophagy, and lipolysis genes were analyzed using Western blot and qPCR. Bioinformatics analysis predicted miR-155-5p's target gene PICALM, confirmed by dual luciferase reporter assay and transfection of miR-155-5p mimic/inhibitor into senescent hepatocytes. RESULTS: Senescent markers (p21, p16, and p-P53) and miR-155-5p were up-regulated in aging liver tissues and senescent hepatocytes. Bioinformatics analysis identified PICALM as a target gene of miR-155-5p, a finding further supported by dual luciferase reporter assays. Inhibition of miR-155-5p reduced expression of senescent marker genes (p16, p21, p-P53), improved autophagy (evidenced by increased LC3B-II and ATG5, and decreased P62), and enhanced lipolysis (indicated by increased ATGL and p-HSL) in senescent hepatocytes. Oil red O staining confirmed that miR-155-5p inhibition significantly reduced lipid accumulation in these cells. CONCLUSIONS: This study suggests a potential new therapeutic approach for age-related hepatic steatosis through the inhibition of miR-155-5p to enhance autophagy.

Autophagy and extracellular vesicles in the liver endothelium: friends or foes?

Role of autophagy in liver sinusoidal endothelial cells (LSECs) and their interaction with hepatic stellate cells (HSC). EndoMT endothelial to mesenchymal transition, EV extracellular vesicle, HSC hepatic stellate cells, LSECs liver sinusoidal endothelial cells, MVB multivesicular body, NO nitric oxide.

Liver histology and hepatic progenitor cell activity in pediatric acute liver failure: Implications for clinical outcome.

BACKGROUND: Hepatic progenitor cell (HPC) activity and regenerative process that follows pediatric acute liver failure (PALF) is still not well understood. This clinicopathological study was thus conducted with an aim to study the correlation of liver histology and HPC activity with outcomes in PALF. METHODS: All PALF patients with available hepatic histological specimens were included and specimens were analyzed for hepatocyte loss, HPC activity [using cytokeratin (CK) 7, CK19, sex-determining region Y-related high mobility group box(SOX)9 and epithelial cell adhesion molecule (EpCAM)], hepatocyte proliferation (using Ki67), and hepatocyte senescence (using p53 and p21). RESULTS: Ninety-four children were included: 22 (23.4%) survived with native liver (SNL) (i.e., the good outcome group) while rest (i.e., the poor outcome group) either died [33%, 35.1%] or received liver transplant (LT) [39%, 41.5%]. When compared to subjects with poor outcomes, those in the SNL group exhibited significantly less severe hepatocyte loss, fewer HPC/hpf, more proliferating hepatocytes, and less senescent hepatocytes (p < .05). Increasing severity of hepatocyte loss (adjusted OR: 9.95, 95% CI: 4.22-23.45, p < .001) was identified as an independent predictor of poor outcome. Eighty percent children with >50% native hepatocyte loss had poor outcome within 10 days of hospitalization. CONCLUSION: In PALF, more severe hepatocyte loss, higher number of HPC activation, lesser number of proliferating hepatocytes, and greater number of senescent hepatocytes are associated with a poor outcome. Loss of >50% hepatocytes is an independent predictor of poor outcome in PALF.

A Single Preservation Solution for Static Cold Storage and Hypothermic Oxygenated Perfusion of Marginal Liver Grafts: A Preclinical Study.

BACKGROUND: Hypothermic oxygenated perfusion (HOPE) improves outcomes of marginal liver grafts. However, to date, no preservation solution exists for both static cold storage (SCS) and HOPE. METHODS: After 30 min of asystolic warm ischemia, porcine livers underwent 6 h of SCS followed by 2 h of HOPE. Liver grafts were either preserved with a single preservation solution (IGL2) designed for SCS and HOPE (IGL2-Machine Perfusion Solution [MPS] group, n = 6) or with the gold-standard University of Wisconsin designed for for SCS and Belzer MPS designed for HOPE (MPS group, n = 5). All liver grafts underwent warm reperfusion with whole autologous blood for 2 h, and surrogate markers of hepatic ischemia-reperfusion injury (IRI) were assessed in the hepatocyte, cholangiocyte, vascular, and immunological compartments. RESULTS: After 2 h of warm reperfusion, livers in the IGL2-MPS group showed no significant differences in transaminase release (aspartate aminotransferase: 65.58 versus 104.9 UI/L/100 g liver; P = 0.178), lactate clearance, and histological IRI compared with livers in the MPS group. There were no significant differences in biliary acid composition, bile production, and histological biliary IRI. Mitochondrial and endothelial damage was also not significantly different and resulted in similar hepatic inflammasome activation. CONCLUSIONS: This preclinical study shows that a novel IGL2 allows for the safe preservation of marginal liver grafts with SCS and HOPE. Hepatic IRI was comparable with the current gold standard of combining 2 different preservation solutions (University of Wisconsin + Belzer MPS). These data pave the way for a phase I first-in-human study and it is a first step toward tailored preservation solutions for machine perfusion of liver grafts.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

Arginase-1 is More Specific Than Hepatocyte Paraffin 1 for Differentiating Hepatocellular Carcinomas With Cytoplasmic Clearing from Nonhepatocellular Clear Cell Tumors in Liver Biopsies.

Arginase-1 (Arg1) and hepatocyte paraffin antigen 1 (HepPar1) are specific and sensitive markers of hepatocellular differentiation. HepPar1 is a granular cytoplasmic immunostain that may be negative in hepatocellular carcinoma (HCC) with cytoplasmic clearing. Arg1 shows uniform cytoplasmic positivity and frequent nuclear positivity. This study was undertaken to determine the staining pattern of Arg1 in HCC with cytoplasmic clearing and compare its use to HepPar1. Fifteen resected HCCs with cytoplasmic clearing and 31 biopsies of clear cell liver tumors (14 HCCs and 17 nonhepatocellular tumors) were identified. Resections were stained with Arg1 to characterize the pattern, intensity, and extent of Arg1 positivity. Biopsies were stained with Arg1 (n=31) and HepPar1 (n=28). In all, 13/15 resected and 11/14 biopsied HCCs with cytoplasmic clearing showed nuclear positivity for Arg1. Both Arg1 and HepPar1 stained significantly more HCCs than nonhepatocellular tumors (13/14 and 11/12, respectively, with P <0.0001 and P =0.0018, respectively). However, HepPar1 stained significantly more nonhepatocellular tumors (5/12) than Arg1 (0/17, P =0.0445). Arg1 frequently displayed nuclear positivity, and interobserver agreement was better for Arg1 ( K =0.93 vs. 0.79). Overall, Arg1 is more specific than HepPar1 for differentiating HCC with cytoplasmic clearing from nonhepatocellular clear cell tumors in the liver. Its staining characteristics, including nuclear positivity, make it easier to interpret in combination with morphology, improving interobserver variability, and it stains significantly fewer mimics than HepPar1.

Discrimination of early HCC using single cell nucleus image and visualization of feature distribution in whole slide images.

Among hepatocellular carcinoma (HCC), early HCC such as well-differentiated hepatocellular carcinoma is more difficult to distinguish from non-cancer than other cancers. In particular, very well-differentiated hepatocellular carcinoma is even more difficult to distinguish, and it is difficult for pathologists to distinguish between cancer and non-cancer from a single nucleus image. If a function to distinguish cancer with a single cell nucleus image is realized, it may be possible to find new features related to nuclei that are useful for differentiating early HCC. The function will also be very helpful in needle biopsy where the area that can be observed is limited.In this study, we investigated the potential to discriminate cancer/non-cancer from an image of a single hepatocyte nucleus using CNN. The results indicated that discrimination was achievable with a correct rate of around 70%.The probability of cancer/non-cancer was visualized on WSI. The visualization results indicated a difference between cancerous and non-cancerous areas in 71% of the cases, which will help pathologists distinguish region of interest. Grouping sections with similar features also proved useful in improving accuracy and visualization results.

Rictor mediates p53 deactivation to facilitate the malignant transformation of hepatocytes and promote hepatocarcinogenesis.

BACKGROUND: Mutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear. METHODS: Microarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo. RESULTS: Our study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P < 0.05). In mice, xenograft tumors overexpressing miR-192 exhibited lower Rictor expression levels, leading to higher p53 activity, and these tumors displayed slower growth compared to untreated HCC cells. CONCLUSIONS: Rictor dynamically shuttles between the nucleus and cytoplasm during HCC development. Its pivotal oncogenic role involves binding and inhibiting p53wt activity within the nucleus in early hepatocarcinogenesis. Targeting Rictor presents a promising strategy for HCC based on p53 status.

Heme Oxygenase-1 Alleviates Ischemia-Reperfusion Injury by Inhibiting Hepatocyte Pyroptosis after Liver Transplantation in Rats.

OBJECTIVE: Heme oxygenase-1 (HO-1) is a protein involved in the inflammatory response following ischemia-reperfusion injury (IRI). Evidence suggests that pyroptosis plays an important role in IRI. However, the underlying mechanism between HO-1 and pyroptosis in IRI requires further investigation. METHODS: Using the "two-cuff" method, a Sprague Dawley rat model of liver transplantation (LT) was established using livers from donors after circulatory death. An automatic biochemical analyzer was used to detect serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and evaluate liver function. Paraffin sections of the rat liver were stained with hematoxylin-eosin (HE) to observe the degree of pathological damage. An enzyme-linked immunosorbent assay was used to detect serum levels of interleukin (IL)-1ß and IL-18. Moreover, western blotting was used to analyze the expression of HO-1, pro-caspase-1, p22, full-gasdermin D (GSDMD), and cleaved-N-GSDMD in the liver. Immunohistochemistry was used to detect NLRP3 expression. RESULTS: HO-1 expression was time-dependent with IRI. HE staining and Suzuki score showed that necrosis was more severe at 6 h after IRI than in controls. Reactive oxygen species (ROS), ALT, and AST levels in the reperfusion were significantly higher at 6 h after IRI. Similar to HO-1 expression, pro-caspase-1, p22, and GSDMD expression in the reperfusion was time-dependent and was significantly higher at 6 h. Compared with the HO-1-shRNA (short hairpin RNA) group, the HO-1 overexpression group significantly inhibited ROS, p22, GSDMD, IL-1ß, IL-18, ALT, and AST. Immunohistochemistry revealed that NLRP3 levels were the highest in the HO-1 overexpression group. CONCLUSIONS: HO-1 improved the survival rate and IRI recovery after LT in rats. This study demonstrates that HO-1 inhibits hepatocyte pyroptosis, thereby reducing IRI after LT.

Histomolecular characterisation of hepatitis B virus induced liver cancer.

Hepatitis B virus (HBV)-associated liver cancer is the third most prevalent cancer-related cause of death worldwide. Different studies have been done on the histomolecular analysis of HBV induced-liver cancer including epigenetics which are dynamic molecular mechanisms to control gene expression without altering the host deoxyribonucleic acid, genomics characterise the integration of the viral genome with host genome, proteomics characterise how gene modifies and results overexpression of proteins, glycoproteomics discover different glyco-biomarker candidates and show glycosylation in malignant hepatocytes, metabolomics characterise how HBV impairs a variety of metabolic functions during hepatocyte immortalisation, exosomes characterise immortalised liver cells in terms of their differentiation and proliferation, and autophagy plays a role in the development of hepatocarcinogenesis linked to HBV infection.