Inhibition of GLUD1 mediated by LASP1 and SYVN1 contributes to hepatitis B virus X protein-induced hepatocarcinogenesis.

Glutamate dehydrogenase 1 (GLUD1) is implicated in oncogenesis. However, little is known about the relationship between GLUD1 and hepatocellular carcinoma (HCC). In the present study, we demonstrated that the expression levels of GLUD1 significantly decreased in tumors, which was relevant to the poor prognosis of HCC. Functionally, GLUD1 silencing enhanced the growth and migration of HCC cells. Mechanistically, the upregulation of interleukin-32 through AKT activation contributes to GLUD1 silencing-facilitated hepatocarcinogenesis. The interaction between GLUD1 and AKT, as well as α-ketoglutarate regulated by GLUD1, can suppress AKT activation. In addition, LIM and SH3 protein 1 (LASP1) interacts with GLUD1 and induces GLUD1 degradation via the ubiquitin-proteasome pathway, which relies on the E3 ubiquitin ligase synoviolin (SYVN1), whose interaction with GLUD1 is enhanced by LASP1. In hepatitis B virus (HBV)-related HCC, the HBV X protein (HBX) can suppress GLUD1 with the participation of LASP1 and SYVN1. Collectively, our data suggest that GLUD1 silencing is significantly associated with HCC development, and LASP1 and SYVN1 mediate the inhibition of GLUD1 in HCC, especially in HBV-related tumors.

Hepatitis B virus core protein stabilizes RANGAP1 to upregulate KDM2A and facilitate hepatocarcinogenesis.

PURPOSE: As a vital component of the hepatitis B virus (HBV) nucleocapsid, HBV core protein (HBC) contributes to hepatocarcinogenesis. Here, we aimed to assess the effects of RANGAP1 and KDM2A on tumorigenesis induced by HBC. METHODS: Co-immunoprecipitation (Co-IP) combined with mass spectrometry were utilized to identify the proteins with the capacity to interact with HBC. The gene and protein levels of RANGAP1 and KDM2A in hepatocellular carcinoma (HCC) and HBV-positive HCC tissues were evaluated using different cohorts. The roles of RANGAP1 and KDM2A in HCC cells mediated by HBC were investigated in vitro and in vivo. Co-IP and western blot were used to estimate the interaction of HBC with RANGAP1 and KDM2A and assess RANGAP1 stabilization regulated by HBC. RESULTS: We discovered that HBC could interact with RANGAP1 and KDM2A, the levels of which were markedly elevated in HCC tissues. Relying on RANGAP1 and KDM2A, HBC facilitated HCC cell growth and migration. The increased stabilization of RANGAP1 mediated by HBC was relevant to the disruption of the interaction between RANGAP1 and an E3 ligase SYVN1. RANGAP1 interacted with KDM2A, and it further promoted KDM2A stabilization by disturbing the interaction between KDM2A and SYVN1. HBC enhanced the interaction of KDM2A with RANGAP1 and upregulated the expression of KDM2A via RANGAP1 in HCC cells. CONCLUSIONS: These findings demonstrate a novel mechanism by which HBC facilitates hepatocarcinogenesis. RANGAP1 and KDM2A could act as potential molecular targets for treating HBV-associated malignancy.

Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production.

BACKGROUND: Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. METHODS: Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. RESULTS: Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. CONCLUSIONS: Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.

Bioinformatics analysis of the proteins interacting with LASP-1 and their association with HBV-related hepatocellular carcinoma.

LIM and SH3 domain protein (LASP-1) is responsible for the development of several types of human cancers via the interaction with other proteins; however, the precise biological functions of proteins interacting with LASP-1 are not fully clarified. Although the role of LASP-1 in hepatocarcinogenesis has been reported, the implication of LASP-1 interactors in HBV-related hepatocellular carcinoma (HCC) is not clearly evaluated. We obtained information regarding LASP-1 interactors from public databases and published studies. Via bioinformatics analysis, we found that LASP-1 interactors were related to distinct molecular functions and associated with various biological processes. Through an integrated network analysis of the interaction and pathways of LASP-1 interactors, cross-talk between different proteins and associated pathways was found. In addition, LASP-1 and several its interactors are significantly altered in HBV-related HCC through microarray analysis and could form a complex co-expression network. In the disease, LASP-1 and its interactors were further predicted to be regulated by a complex interaction network composed of different transcription factors. Besides, numerous LASP-1 interactors were associated with various clinical factors and related to the survival and recurrence of HBV-related HCC. Taken together, these results could help enrich our understanding of LASP-1 interactors and their relationships with HBV-related HCC.

Bioinformatics Analysis Reveals Distinct Molecular Characteristics of Hepatitis B-Related Hepatocellular Carcinomas from Very Early to Advanced Barcelona Clinic Liver Cancer Stages.

Hepatocellular carcinoma (HCC)is the fifth most common malignancy associated with high mortality. One of the risk factors for HCC is chronic hepatitis B virus (HBV) infection. The treatment strategy for the disease is dependent on the stage of HCC, and the Barcelona clinic liver cancer (BCLC) staging system is used in most HCC cases. However, the molecular characteristics of HBV-related HCC in different BCLC stages are still unknown. Using GSE14520 microarray data from HBV-related HCC cases with BCLC stages from 0 (very early stage) to C (advanced stage) in the gene expression omnibus (GEO) database, differentially expressed genes (DEGs), including common DEGs and unique DEGs in different BCLC stages, were identified. These DEGs were located on different chromosomes. The molecular functions and biology pathways of DEGs were identified by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the interactome networks of DEGs were constructed using the NetVenn online tool. The results revealed that both common DEGs and stage-specific DEGs were associated with various molecular functions and were involved in special biological pathways. In addition, several hub genes were found in the interactome networks of DEGs. The identified DEGs and hub genes promote our understanding of the molecular mechanisms underlying the development of HBV-related HCC through the different BCLC stages, and might be used as staging biomarkers or molecular targets for the treatment of HCC with HBV infection.

CD4+CXCR5+ T cells activate CD27+IgG+ B cells via IL-21 in patients with hepatitis C virus infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG+ B cells in patients with chronic hepatitis C (CHC) infection have not been thoroughly elucidated. CD4+CXCR5+ follicular helper T (Tfh) cells, via interleukin (IL)-21 secretion, activate B cells. However, the role of CD4+CXCR5+ T cells in the activation of IgG+ B cells in CHC patients is not clear. METHODS: The frequency of IgG+ B cells, including CD27-IgG+ B and CD27+IgG+ B cells, the expression of the activation markers (CD86 and CD95) in IgG+ B cells, and the percentage of circulating CD4+CXCR5+ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The concentrations of serum IL-21 were analyzed using ELISA. The role of CD4+CXCR5+ T cells in the activation of IgG+ B cells was investigated using a co-culture system. RESULTS: A significantly lower proportion of CD27+IgG+ B cells with increased expression of CD86 and CD95 was observed in CHC patients. The expression of CD95 was negatively correlated with the percentage of CD27+IgG+ B cells, and it contributed to CD27+IgG+ B cell apoptosis. Circulating CD4+CXCR5+ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4+CXCR5+ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27+IgG+ B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27+IgG+ B cells. CONCLUSIONS: HCV infection increased the frequency of CD4+CXCR5+ T cells and decreased the frequency of CD27+IgG+ B cells. CD4+CXCR5+ T cells activated CD27+IgG+ B cells via the secretion of IL-21.

Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients

Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expen- sive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen) had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%), and hepatitis C virus core antigen decrease greater than 1 log10 IU/mL (early virological response based on hepatitis C virus core antigen) had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.Z%). Analysis on the validation group demonstrated a positive predictivevalue of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to hepatitis C virus RNA in predicting sustained virologic response of chronic genotype 1 hepatitis C virus infected patients, and can be used to guide anti-hepatitis C virus treatment, especially in resource-limited areas.

Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients.

Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expensive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen) had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%), and hepatitis C virus core antigen decrease greater than 1log10IU/mL (early virological response based on hepatitis C virus core antigen) had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.2%). Analysis on the validation group demonstrated a positive predictive value of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to hepatitis C virus RNA in predicting sustained virologic response of chronic genotype 1 hepatitis C virus infected patients, and can be used to guide anti-hepatitis C virus treatment, especially in resource-limited areas.

Hepatitis C virus core antigen, an earlier and stronger predictor on sustained virological response in patients with genotype 1 HCV infection.

BACKGROUND: Earlier kinetics of serum HCV core antigen (HCVcAg) and its predictive value on sustained virological response (SVR) were investigated in patients with genotype 1 HCV infection during antiviral treatment. METHODS: In a multi-centered, randomized and positive drug-controlled phase IIb clinical trial on type Y peginterferon α-2b ( NCT01140997), forty-eight CHC patients who participated in pharmacokinetics were randomly divided into 4 cohorts and treated with PegIFNα (type Y peginterferon α-2b 90 µg, 135 µg, 180 µg and PegIFNα-2a 180 µg, respectively, once a week) and ribavirin (< 75 kg, 1000 mg daily and ≥ 75 kg, 1200 mg daily) for 48 weeks, and then followed up for 24 weeks. 32 patients infected with genotype 1 HCV and completed the whole process were included in this study. HCV RNAs were detected at baseline, and weeks 4, 12, 24, 48 and 72 using Cobas TaqMan. ARCHITECT HCVcAg was performed at 24, 48, 72, 96, 120 and 144 h in addition to the above time points. The receiver operating curves (ROCs) were performed to study the predictive values of HCVcAg decline on SVR. RESULTS: Following antiviral treatment, serum HCVcAg levels rapidly declined within the first week and correlated well with corresponding HCV RNA at baseline, weeks 4, 12, 24, 48 and 72 (rs = 0.969, 0.928, 0.999, 0.983, 0.985 and 0.946, respectively, P < 0.001). All of the areas under the receiver operating curves (AUROCs) were more than 0.80 and showed good predictive power on SVR at 24, 48, 72, 96, 120 and 144 h. The144 h was the best predictive time point of HCVcAg decline on SVR because of its largest AUROC (more than 0.90). CONCLUSIONS: Early kinetics of serum HCVcAg predicts SVR very well in genotype 1 CHC patients during antiviral treatment, and its reduction value at 144 h is an earlier and stronger predictor on SVR than rapid virological response and early virological response. (TRN: NCT01140997).

HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module.

AIM: To investigate the possible mechanism by which hepatitis B virus X protein (HBx) mediates apoptosis of HepG2 cells. METHODS: HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high-expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX (HBx antagonist) were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases (MAPKs), were measured in each group. RESULTS: Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and JNKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and JNKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. CONCLUSION: HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.