Eleven metabolism­related genes composed of Stard5 predict prognosis and contribute to EMT phenotype in HCC.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with a high mortality and poor survival rate. Abnormal tumor metabolism is considered a hallmark of HCC and is a potential therapeutic target. This study aimed to identify metabolism-related biomarkers to evaluate the prognosis of patients with HCC. METHOD: The Cancer Genome Atlas (TCGA) database was used to explore differential metabolic pathways based on high and low epithelial-mesenchymal transition (EMT) groupings. Genes in differential metabolic pathways were obtained for HCC metabolism-related molecular subtype analysis. Differentially expressed genes (DEGs) from the three subtypes were subjected to Lasso Cox regression analysis to construct prognostic risk models. Stard5 expression in HCC patients was detected by western blot and immunohistochemistry (IHC), and the role of Stard5 in the metastasis of HCC was investigated by cytological experiments. RESULTS: Unsupervised clustering analysis based on metabolism-related genes revealed three subtypes in HCC with differential prognosis. A risk prognostic model was constructed based on 11 genes (STARD5, FTCD, SCN4A, ADH4, CFHR3, CYP2C9, CCL14, GADD45G, SOX11, SCIN, and SLC2A1) obtained by LASSO Cox regression analysis of the three subtypes of DEGs. We validated that the model had a good predictive power. In addition, we found that the high-risk group had a poor prognosis, higher proportion of Tregs, and responded poorly to chemotherapy. We also found that Stard5 expression was markedly decreased in HCC tissues, which was associated with poor prognosis and EMT. Knockdown of Stard5 contributed to the invasion and migration of HCC cells. Overexpression of Stard5 inhibited EMT in HCC cells. CONCLUSION: We developed a new model based on 11 metabolism-related genes, which predicted the prognosis and response to chemotherapy or immunotherapy for HCC. Notably, we demonstrated for the first time that Stard5 acted as a tumor suppressor by inhibiting metastasis in HCC.

Six immune-related promising biomarkers may promote hepatocellular carcinoma prognosis: a bioinformatics analysis and experimental validation.

BACKGROUND: Abnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear. METHODS: Differentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis. RESULTS: We identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression. CONCLUSIONS: Briefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression.

The long non-coding RNA DKFZp434J0226 regulates the alternative splicing process through phosphorylation of SF3B6 in PDAC.

BACKGROUND: Long noncoding RNAs (lncRNAs), a type of pervasive genes that regulates various biological processes, are differentially expressed in different types of malignant tumors. The role of lncRNAs in the carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we investigated the role of the lncRNA DKFZp434J0226 in PDAC. METHODS: Aberrantly expressed mRNAs and lncRNAs among six PDAC and paired non-tumorous tissues were profiled using microarray analysis. Quantitative real-time polymerase chain reaction was used to evaluate DKFZp434J0226 expression in PDAC tissues. CCK-8 assay, wound-healing assay, soft agar colony formation assay, and transwell assay were performed to assess the invasiveness and proliferation of PDAC cells. Furthermore, RNA pull-down, immunofluorescence, RNA immunoprecipitation, and western blotting assays were performed to investigate the association between DKFZp434J0226 and SF3B6. Tumor xenografts in mice were used to test for tumor formation in vivo. RESULTS: In our study, 222 mRNAs and 128 lncRNAs were aberrantly expressed (≥ twofold change). Of these, 66 mRNAs and 53 lncRNAs were upregulated, while 75 lncRNAs and 156 mRNAs were downregulated. KEGG pathway analysis and the Gene ontology category indicated that these genes were associated with the regulation of mRNA alternative splicing and metabolic balance. Clinical analyses revealed that overexpression of DKFZp434J0226 was associated with worse tumor grading, frequent perineural invasion, advanced tumor-node-metastasis stage, and decreased overall survival and time to progression. Functional assays demonstrated that DKFZp434J0226 promoted PDAC cell migration, invasion, and growth in vitro and accelerated tumor proliferation in vivo. Mechanistically, DKFZp434J0226 interacted with the splicing factor SF3B6 and promoted its phosphorylation, which further regulated the alternative splicing of pre-mRNA. CONCLUSIONS: This study indicates that DKFZp434J0226 regulates alternative splicing through phosphorylation of SF3B6 in PDAC and leads to an oncogenic phenotype in PDAC.

The role of Sphingomyelin synthase 2 (SMS2) in platelet activation and its clinical significance.

BACKGROUND: Sphingomyelin (SM) is an essential component of biological lipid rafts, and it plays an indispensable role in maintaining plasma membrane stability and in mediating signal transduction. The ultimate biosynthesis of SM is catalyzed by two sphingomyelin synthases (SMSs) namely SMS1 and SMS2, which are selectively distributed in the trans-Golgi apparatus and the plasma membrane. It has been demonstrated that SMS2 acts as an irreplaceable molecule in the regulation of transmembrane signaling, and loss of SMS2 has been reported to worsen atherosclerosis and liver steatosis. However, the function of SMS2 in platelet activation and its association with the pathological process of thrombosis in acute coronary syndrome (ACS) and portal hypertension (PH) remain unclear. METHODS: In this study, we tested the role of SMS2 in platelet activation and thrombosis using SMS2 knockout (SMS2 -/-) mice and SMS2-specific inhibitor, D609. Furthermore, we detected SMS2 expression in patients with ACS and PH. RESULTS: SMS2 -/- platelets showed significant reduction in platelet aggregation, spreading, clot retraction and in vivo thrombosis. Similar inhibitory effects on platelet activation were detected in D609-treated wild-type platelets. PLCγ/PI3K/Akt signaling pathway was inhibited in SMS2 -/- platelets and D609-treated wild-type platelets. In addition, we discovered that platelet SMS2 expression was remarkably increased in patients with ACS and PH, compared with healthy subjects. CONCLUSIONS: Our study indicates that SMS2 acts as a positive regulator of platelet activation and thrombosis, and provides a theoretical basis for the potential use of D609 in anti-thrombosis treatment.

Smurf2, an E3 ubiquitin ligase, interacts with PDE4B and attenuates liver fibrosis through miR-132 mediated CTGF inhibition.

We previously reported that Smad ubiquitin regulatory factor 2 (Smurf2) activity was decreased in human fibrotic livers. Here, we overexpressed Smurf2 in livers of transgenic mice and observed inhibited collagen deposition and hepatic stellate cell activation in fibrotic model induced by carbon tetrachloride treatment or bile duct ligation. Hepatic Smurf2 overexpression also inhibited the production of connective tissue growth factor (CTGF), a central mediator of liver fibrosis. Using miRNA array and bioinformatics analyses, we identified miR-132 as a mediator of this inhibitory effect. miR-132 directly targets the 3'-untranslated region of CTGF and was transcriptionally upregulated by cAMP-PKA-CREB signaling. In addition, Smurf2 activated cAMP-PKA-CREB pathway by interacting with phosphodiesterase 4B (PDE4B) and facilitating its degradation. Thus, we have demonstrated a previously unrecognized anti-fibrotic pathway controlled by Smurf2.

Cholestasis-induced bile acid elevates estrogen level via farnesoid X receptor-mediated suppression of the estrogen sulfotransferase SULT1E1.

The liver is the main site of estrogen metabolism, and liver disease is usually associated with an abnormal estrogen status. However, little is known about the mechanism underlying this connection. Here, we investigated the effects of bile acid (BA)-activated farnesoid X receptor (FXR) on the metabolism of 17ß-estradiol (E2) during blockage of bile flow (cholestasis). Correlations between BA levels and E2 concentrations were established in patients with cholestasis, and hepatic expression profiles of key genes involved in estrogen metabolism were investigated in both WT and FXR-/- mice. We found that the elevated E2 level positively correlated with BA concentrations in the patients with cholestasis. We further observed that bile duct ligation (BDL) increases E2 levels in mouse serum, and this elevation effect was alleviated by deleting the FXR gene. Of note, FXR down-regulated the expression of hepatic sulfotransferase SULT1E1, the primary enzyme responsible for metabolic estrogen inactivation. At the molecular level, we found that FXR competes with the protein acetylase CREB-binding protein (CBP) for binding to the transcription factor hepatocyte nuclear factor 4α (HNF4α). This competition decreased HNF4α acetylation and nuclear retention, which, in turn, repressed HNF4α-dependent SULT1E1 gene transcription. These findings suggest that cholestasis induces BA-activated FXR activity, leading to downstream inhibition of SULT1E1 and hence impeding hepatic degradation of estrogen.

Spermine oxidase is upregulated and promotes tumor growth in hepatocellular carcinoma.

AIM: The polyamine catabolic enzyme, spermine oxidase (SMOX) is upregulated in chronic inflammatory conditions and linked to increased reactive oxygen species and DNA damage in various forms of cancers. The present study aims to explore the expression pattern and biological function of SMOX in hepatocellular carcinoma (HCC). METHODS: We used quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry to examine SMOX expression in four HCC cell lines and 120 HCC clinical samples, and the clinical significance of SMOX was analyzed. The biological function of SMOX on HCC cells was detected both in vitro and in vivo. RESULTS: Results showed that SMOX was overexpressed in HCC cell lines and clinical HCC tissues. Moreover, SMOX expression levels were gradually increased in normal liver, chronic hepatitis, and HCC tissues. Increased SMOX expression was correlated with poor clinical features of HCC. Patients with positive SMOX expression in tumor tissues indicated worse overall survival (P = 0.008) and shorter relapse-free survival (P = 0.002). Knockdown of SMOX inhibited HCC cell proliferation, arrested cell cycle at S phase, and resulted in an increase of apoptosis. The in vivo study showed that inhibition of SMOX in HCC cells significantly repressed tumor growth in nude mice. Furthermore, we showed that SMOX might exert its function by regulating the phosphatidylinositol 3'-kinase/protein kinase B signaling pathway. CONCLUSION: Our data indicated that SMOX upregulation could be a critical oncogene in HCC and might serve as a valuable prognostic marker and potential therapeutic target for HCC.

BH3 Mimetic ABT-199 Enhances the Sensitivity of Gemcitabine in Pancreatic Cancer in vitro and in vivo.

BACKGROUND AND AIMS: Pancreatic cancer is an aggressive malignancy with poor prognosis. Gemcitabine is the standard chemotherapeutic drug used to treat the disease; however, it has a low response rate. Therefore, there is an urgent need to develop new and safe therapies to enhance sensitivity to gemcitabine in treating pancreatic cancer. METHODS: The synergistic effect of gemcitabine combined with specific B cell CLL/lymphoma 2 (Bcl-2) inhibitor ABT-199 against pancreatic cancer was tested using cell viability, cell cycle, and apoptosis assays in vitro and in an MIA Paca-2 xenograft model in vivo. Its underlying mechanism was explored using western blotting analysis of Bcl-2 family proteins. RESULTS: ABT-199 not only enhanced the effect of gemcitabine on cell growth inhibition but also promoted gemcitabine-induced apoptosis in pancreatic cancer cell lines. Gemcitabine decreased the expression of anti-apoptotic protein Mcl-1 but increased the expression of anti-apoptotic protein Bcl-2. ABT-199 downregulated the gemcitabine-induced production of Bcl-2 and increased the expression of pro-apoptotic protein Bcl-2 interacting protein (BIM). Mouse xenograft experiments also confirmed the synergistic effect of gemcitabine and ABT-199 on tumor growth inhibition and the induction of tumor cell apoptosis. CONCLUSION: Our results indicated that ABT-199 improved the anti-tumor effect of gemcitabine on pancreatic cancer by downregulating gemcitabine-induced overexpression of Bcl-2. ABT-199 has already been investigated in phase 3 clinical trials for chronic lymphocytic leukemia; therefore, it may serve as a potential drug to improve the sensitivity of pancreatic cancer to gemcitabine.

Prp19 Arrests Cell Cycle via Cdc5L in Hepatocellular Carcinoma Cells.

Pre-mRNA processing factor 19 (Prp19) is involved in many cellular events including pre-mRNA processing and DNA damage response. Recently, it has been identified as a candidate oncogene in hepatocellular carcinoma (HCC). However, the role of Prp19 in tumor biology is still elusive. Here, we reported that Prp19 arrested cell cycle in HCC cells via regulating G2/M transition. Mechanistic insights revealed that silencing Prp19 inhibited the expression of cell division cycle 5-like (Cdc5L) via repressing the translation of Cdc5L mRNA and facilitating lysosome-mediated degradation of Cdc5L in HCC cells. Furthermore, we found that silencing Prp19 induced cell cycle arrest could be partially resumed by overexpressing Cdc5L. This work implied that Prp19 participated in mitotic progression and thus could be a promising therapeutic target of HCC.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway.

Aberrant activation of Notch signaling frequently occurs in liver cancer, and is associated with liver malignancies. However, the mechanisms regulating pathologic Notch activation in hepatocellular carcinoma (HCC) remain unclear. Protein O-fucosyltransferase 1 (Pofut1) catalyzes the addition of O-linked fucose to the epidermal growth factor-like repeats of Notch. In the present study, we detected the expression of Pofut1 in 8 HCC cell lines and 253 human HCC tissues. We reported that Pofut1 was overexpressed in HCC cell lines and clinical HCC tissues, and Pofut1 overexpression clinically correlated with the unfavorable survival and high disease recurrence in HCC. The in vitro assay demonstrated that Pofut1 overexpression accelerated the cell proliferation and migration in HCC cells. Furthermore, Pofut1 overexpression promoted the binding of Notch ligand Dll1 to Notch receptor, and hence activated Notch signaling pathway in HCC cells, indicating that Pofut1 overexpression could be a reason for the aberrant activation of Notch signaling in HCC. Taken together, our findings indicated that an aberrant activated Pofut1-Notch pathway was involved in HCC progression, and blockage of this pathway could be a promising strategy for the therapy of HCC.

CD86⁺/CD206⁺, Diametrically Polarized Tumor-Associated Macrophages, Predict Hepatocellular Carcinoma Patient Prognosis.

Tumor-associated macrophages (TAMs), the most abundant infiltrating immune cells in tumor microenvironment, have distinct functions in hepatocellular carcinoma (HCC) progression. CD68⁺ TAMs represent multiple polarized immune cells mainly containing CD86⁺ antitumoral M1 macrophages and CD206⁺ protumoral M2 macrophages. TAMs expression and density were assessed by immunohistochemical staining of CD68, CD86, and CD206 in tissue microarrays from 253 HCC patients. Clinicopathologic features and prognostic value of these markers were evaluated. We found that CD68⁺ TAMs were not associated with clinicopathologic characteristics and prognosis in HCC. Low presence of CD86⁺ TAMs and high presence of CD206⁺ TAMs were markedly correlated with aggressive tumor phenotypes, such as multiple tumor number and advanced tumor-node-metastasis (TNM) stage; and were associated with poor overall survival (OS) (p = 0.027 and p = 0.024, respectively) and increased time to recurrence (TTR) (p = 0.037 and p = 0.031, respectively). In addition, combined analysis of CD86 and CD206 provided a better indicator for OS (p = 0.011) and TTR (p = 0.024) in HCC than individual analysis of CD86 and CD206. Moreover, CD86⁺/CD206⁺ TAMs predictive model also had significant prognosis value in α-fetoprotein (AFP)-negative patients (OS: p = 0.002, TTR: p = 0.005). Thus, these results suggest that combined analysis of immune biomarkers CD86 and CD206 could be a promising HCC prognostic biomarker.

CAPS1 Negatively Regulates Hepatocellular Carcinoma Development through Alteration of Exocytosis-Associated Tumor Microenvironment.

The calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles (DCVs) in neurons and neuroendocrine cells. The role of CAPS1 in cancer biology remains unknown. The purpose of this study was to investigate the role of CAPS1 in hepatocellular carcinoma (HCC). We determined the levels of CAPS1 in eight hepatoma cell lines and 141 HCC specimens. We evaluated the prognostic value of CAPS1 expression and its association with clinical parameters. We investigated the biological consequences of CAPS1 overexpression in two hepatoma cell lines in vitro and in vivo. The results showed that loss of CAPS1 expression in HCC tissues was markedly correlated with aggressive tumor phenotypes, such as high-grade tumor node metastasis (TNM) stage (p = 0.003) and absence of tumor encapsulation (p = 0.016), and was associated with poor overall survival (p = 0.008) and high recurrence (p = 0.015). CAPS1 overexpression inhibited cell proliferation and migration by changing the exocytosis-associated tumor microenvironment in hepatoma cells in vitro. The in vivo study showed that CAPS1 overexpression inhibited xenograft tumor growth. Together, these results identified a previously unrecognized tumor suppressor role for CAPS1 in HCC development.

BIRC6 promotes hepatocellular carcinogenesis: interaction of BIRC6 with p53 facilitating p53 degradation.

The genes that encode inhibitor of apoptosis proteins (IAPs) are frequently overexpressed in human cancers. However, the expression pattern and clinical significance of BIRC6, a member of IAPs, in hepatocellular carcinoma (HCC) remains unclear. Here we investigated the role of BIRC6 in hepatocellular carcinogenesis. We used immunoblot and immunochemical analyses to determine the levels of BIRC6 in 7 hepatoma cell lines and 160 HCC specimens. We evaluated the proognostic value of BIRC6 expression and its association with clinical parameters. A lentivirus-mediated silencing method was used to knockdown BIRC6, and the biological consequences of BIRC6 silencing in three hepatoma cell lines were investigated in vitro and in vivo. We found that BIRC6 overexpression was significantly correlated with serum ALT level and HCC vascular invasion. Patients with positive BIRC6 expression in tumor tissue had a poor survival and a high rate of recurrence. BIRC6 knockdown remarkably suppressed cell proliferation, caused G1/S arrest and sensitized hepatoma cells to sorafenib-induced apoptosis in hepatoma cells, which was partly reversed by RNA interference targeting p53. The mechanistic study revealed that BIRC6 interacted with p53 and facilitated its degradation. The in vivo study showed that BIRC6 knockdown inhibited xenograft tumor growth and increased the sensitivity of tumor cells to sorafenib in nude mice. Taken together, these findings demonstrate that BIRC6 overexpression in HCC specimens is indicative of poor prognosis and that its interaction with p53 facilitates the degradation of p53, leading to carcinogenesis and an anti-apoptotic status.

Ubiquitin C-terminal Hydrolase 37, a novel predictor for hepatocellular carcinoma recurrence, promotes cell migration and invasion via interacting and deubiquitinating PRP19.

Ubiquitin C-terminal hydrolase 37 (UCH37) plays a crucial role in numerous biological processes and is also involved in oncogenesis. In this study, clinicopathologic data showed that UCH37 was over-expressed in hepatocellular carcinoma (HCC) cancerous tissues and was a significant predictor for time to recurrence (TTR). In vitro, we discovered that UCH37 could promote cell migration and invasion. Subsequently, we utilized Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in UCH37 over-expressing cells compared with the control cells, and found that PRP19, an essential RNA splicing factor, was up-regulated. The relationship between UCH37, PRP19 and the capability of cell migration and invasion was further confirmed. Collectively, this study demonstrated that UCH37 could promote cell migration and invasion in HCC cell lines through interacting and deubiquitinating PRP19, and suggested that UCH37 could be a novel predictor for HCC recurrence after curative resection.

Activation of farnesoid X receptor (FXR) protects against fructose-induced liver steatosis via inflammatory inhibition and ADRP reduction.

Fructose is a key dietary factor in the development of nonalcoholic fatty liver disease (NAFLD). Here we investigated whether WAY-362450 (WAY), a potent synthetic and orally active FXR agonist, protects against fructose-induced steatosis and the underlying mechanisms. C57BL/6J mice, fed 30% fructose for 8 weeks, were treated with or without WAY, 30 mg/kg, for 20 days. The elevation of serum and hepatic triglyceride in mice fed 30% fructose was reversed by WAY treatment. Histologically, WAY significantly reduced triglyceride accumulation in liver, attenuated microphage infiltration and protected the junction integrity in intestine. Moreover, WAY remarkably decreased portal endotoxin level, and lowered serum TNFα concentration. In lipopolysaccharide (LPS)-induced NAFLD model, WAY attenuated serum TNFα level. Moreover, WAY suppressed LPS-induced expression of hepatic lipid droplet protein adipose differentiation-related protein (ADRP), down-regulation of it in mice fed 30% fructose. Furthermore, WAY repressed lipid accumulation and ADRP expression in a dose-dependent manner in palmitic acid (PA)-treated HepG2 and Huh7 cells. WAY suppressed TNFα-induced ADRP up-regulation via competing with AP-1 for ADRP promoter binding region. Together, our findings suggest that WAY, an FXR agonist, attenuates liver steatosis through multiple mechanisms critically involved in the development of hepatosteatosis, and represents a candidate for NAFLD treatment.

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR(-/-)) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR(-/-) mice fed MCD diet (FXR(-/-)/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR(-/-)/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR(-/-)/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR(-/-)/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.

Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio: Markers predicting immune-checkpoint inhibitor efficacy and immune-related adverse events.

We conducted a comprehensive review of existing prediction models pertaining to the efficacy of immune-checkpoint inhibitor (ICI) and the occurrence of immune-related adverse events (irAEs). The predictive potential of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in determining ICI effectiveness has been extensively investigated, while limited research has been conducted on predicting irAEs. Furthermore, the combined model incorporating NLR and PLR, either with each other or in conjunction with additional markers such as carcinoembryonic antigen, exhibits superior predictive capabilities compared to individual markers alone. NLR and PLR are promising markers for clinical applications. Forthcoming models ought to incorporate established efficacious models and newly identified ones, thereby constituting a multifactor composite model. Furthermore, efforts should be made to explore effective clinical application approaches that enhance the predictive accuracy and efficiency.

Efficient gene delivery by multifunctional star poly (ß-amino ester)s into difficult-to-transfect macrophages for M1 polarization.

Gene delivery to macrophages holds great promise for cancer immunotherapy. However, traditional gene delivery methods exhibit low transfection efficiency in macrophages. The star-shaped topological structure of polymers is known to encapsulate genes inside their cores, thereby facilitating sustained release of the genetic material. Herein, combining the structural advantages of star polymers and the transfection advantages of poly (ß-amino ester)s (PAEs), we developed a novel linear oligomer grafting-onto strategy to synthesize a library of multi-terminal star structured PAEs (SPAEs), and evaluated their gene delivery efficiency in various tissue cells. The transfection with human hepatocellular carcinoma cells (HepG2, HCC-LM3 cells and MHCC-97H cells), rat normal liver cells (BRL-3 A cells), human ovarian cancer cells (A2780 cells), African green monkey kidney cells (Vero cells), human cervical cancer cells (HeLa cells), human chondrosarcoma cells (SW1353 cells), and difficult-to-transfect human epidermal keratinocytes (HaCaT cells) and normal human fibroblast cells (NHF cells) showed that SPAEs exhibited superior transfection profile. The GFP transfection efficiency of top-performing SPAEs in HeLa cells (96.1%) was 2.1-fold, and 3.2-fold higher compared to jetPEI and Lipo3000, respectively, indicating that the star-shaped topological structure can significantly enhance the transfection efficiency of PAEs. More importantly, the top-performing SPAEs could efficiently deliver Nod2 DNA to difficult-to-transfect RAW264.7 macrophages, with a high transfection efficiency of 33.9%, which could promote macrophage M1 polarization and enhanced CD8+ T cell response in co-incubation experiments. This work advances gene therapy by targeting difficult-to-transfect macrophages and remodeling the tumor immune microenvironment.

Inhibiting PLA2G7 reverses the immunosuppressive function of intratumoral macrophages and augments immunotherapy response in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an exceptionally immunosuppressive malignancy characterized by limited treatment options and a dismal prognosis. Macrophages constitute the primary and heterogeneous immune cell population within the HCC microenvironment. Our objective is to identify distinct subsets of macrophages implicated in the progression of HCC and their resistance to immunotherapy. METHODS: Intratumoral macrophage-specific marker genes were identified via single-cell RNA sequencing analyses. The clinical relevance of phospholipase A2 Group VII (PLA2G7), a pivotal enzyme in phospholipid metabolism, was assessed in patients with HCC through immunohistochemistry and immunofluorescence. Flow cytometry and an in vitro co-culture system were used to elucidate the specific role of PLA2G7 in macrophages. Orthotopic and subcutaneous HCC mouse models were employed to evaluate the potential of the PLA2G7 inhibitor in complementing immune checkpoint blockade (ICB) therapy. RESULTS: Single-cell RNA sequencing analyses disclosed predominant PLA2G7 expression in intratumoral macrophages within the HCC microenvironment. The macrophage-specific PLA2G7 was significantly correlated with poorer prognosis and immunotherapy resistance in patients with HCC. PLA2G7high macrophages represent a highly immunosuppressive subset and impede CD8 T-cell activation. Pharmacological inhibition of PLA2G7 by darapladib improved the therapeutic efficacy of anti-programmed cell death protein 1 antibodies in the HCC mouse models. CONCLUSIONS: Macrophage-specific PLA2G7 serves as a novel biomarker capable of prognosticating immunotherapy responsiveness and inhibiting PLA2G7 has the potential to enhance the efficacy of ICB therapy for HCC.