Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20

<div><p>Hepatitis B virus (HBV) infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx) is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD), and the formation of death-inducing signaling complex (DISC), thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.</p></div

HBx enhances the sensitivity of hepatocytes to TRAIL-induced apoptosis.

<p>(A, B) L-O2, L-O2-pCMV, and L-O2-HBx cells were treated with PBS or 30 ng/ml TRAIL. After 24 h, the cells were labeled by Annexin V/PI (A) or TUNEL (B) and examined by flow cytometry.</p

HBx enhances caspase-8 activity by downregulating its A20-mediated K63-linked polyubiquitination.

<p>(A, B) The interactions of DR5 with A20, RIP1, caspase-8, and FADD were detected by co-immunoprecipitation 15 min after treatment with TRAIL (30 ng/ml) in L-O2 and L-O2-HBx cells (A), or in L-O2-HBx cells transfected with the A20 expression vector or control vector (B). (C, D) K63-linked polyubiquitination of caspase-8 was detected after treatment with TRAIL (30 ng/ml) in L-O2 and L-O2-HBx cells (C) or in L-O2-HBx cells transfected with the A20 expression vector or control vector (D). (E) <i>In vitro</i> ubiquitination was carried out in a reaction consisting of the components as indicated (<i>top</i>) with ubiquitinated caspase 8 detected by a HRP linked streptavidin or specific antibodies on IB. (F) Caspase-8 was immunoprecipitated from L-O2, L-O2-HBx cells, or L-O2-HBx cells tranfected with pA20 upon TRAIL treatment, followed by a second immunoprecipitation with an anti-K63 ubiquitin antibody. The ubiquitination of caspase-8 was then detected by immunoblotting. Representative data from at least three independent experiments are shown.</p

HBx regulates miR-125a transcription in a methylation-dependent manner.

<p>(A) L-O2, L-O2-pCMV, and L-O2-HBx cells were analyzed for miR-125a expression by real-time RT-PCR. (B) L-O2-HBx cells were transfected with control siRNA or HBx siRNA, and miR-125a expression was analyzed by real-time RT-PCR. (C) The methylation of miR-125a CpG sites was analyzed in L-O2 and L-O2-HBx cells by bisulfite sequencing analysis. At least five independent clones were sequenced per sample. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. (D) L-O2 cells were analyzed for miR-125a expression by qRT-PCR after treatment with 5 μM 5′-Aza for 72 h. Data are from three independent experiments and are presented as the mean ± SEM (**P<0.01, ***P<0.001).</p

HBx inhibits A20 expression by upregulating miR-125a.

<p>(A) The luciferase activity was measured in 293T cells transfected with miR-125a or the control plasmid (NC), along with reporter plasmids (pMIR-) containing the intact or mutant binding sites at the A20 ORF or UTR. (B) Luciferase activity was measured in 293T cells transfected with the miR-125a inhibitor or the control plasmid (NC), along with the pMIR- constructs as in A. (C) The expression of A20 was detected by western blot in L-O2 cells transfected with miR-125a, anti-miR-125a or their controls. Data are from three independent experiments and are presented as the mean ± SEM. *P<0.05, **P<0.01 compared with the NC controls.</p

HBx inhibits A20 expression in hepatocytes at the protein but not the mRNA level.

<p>(A) L-O2, L-O2-pCMV, and L-O2-HBx cells were analyzed for A20 and HBx expression by western blot. (B) L-O2 cells and HepG2 cells were transfected with the pCMV-HBx plasmid at the indicated dose, and after 48 h, A20 expression was analyzed by western blot. (C) L-O2-HBx cells were transfected with control siRNA or HBx siRNA, and A20 and HBx expression was analyzed by western blot. (D, E) L-O2, L-O2-pCMV, and L-O2-HBx cells were analyzed for A20 mRNA expression by RT-PCR and real-time PCR. (F) HepG2 and HepG2.2.15 cells were analyzed for A20 mRNA expression by quantitative PCR. The results are from three independent experiments and are presented as the mean ± SEM.</p

The transcript miR-125a/A20 mediates HBx-induced modulation of hepatocyte apoptosis.

<p>The apoptotic percentages were examined by flow cytometry 24 h after treatment with TRAIL (30 ng/ml) in (A, B) L-O2 cells transfected with miR-125a or the control, in (C, D) L-O2-HBx cells transfected with anti-miR-125a or the control, or in (E, F) L-O2-HBx cells transfected with the control plasmid or pCMV-A20. Representative data from at least three independent experiments are shown.</p

Additional file 1 of Modulation of alveolar macrophage and mitochondrial fitness by medicinal plant-derived nanovesicles to mitigate acute lung injury and viral pneumonia

Supplementary Material

Integrative Analysis of Multi-Omic Data for the Characteristics of Endometrial Cancer

Endometrial cancer (EC) is a frequently diagnosed gynecologic cancer. Identifying reliable prognostic genes for predicting EC onset is crucial for reducing patient morbidity and mortality. Here, a comprehensive strategy with transcriptomic and proteomic data was performed to measure EC’s characteristics. Based on the publicly available RNA-seq data, death-associated protein kinase 3, recombination signal-binding protein for the immunoglobulin kappa J region, and myosin light chain 9 were screened out as potential biomarkers that affect the EC patients’ prognosis. A linear model was further constructed by multivariate Cox regression for the prediction of the risk of being malignant. From further integrative analysis, exosomes were found to have a highly enriched role that might participate in EC occurrence. The findings were validated by qRT-polymerase chain reaction (PCR) and western blotting. Collectively, we constructed a prognostic-gene-based model for EC prediction and found that exosomes participate in EC incidents, revealing significantly promising support for the diagnosis of EC

MOESM1 of Understanding immune phenotypes in human gastric disease tissues by multiplexed immunohistochemistry

Additional file 1. Supplemental methods, supplementary tables for patients. Figure S1. H&E staining of the two TMAs. Figure S2. Comparison of the results of ImageJ software and the score by pathologists