The oncogenic role of hepatitis B virus X gene in hepatocarcinogenesis: recent updates.

Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancers with high mortality rate. Among its various etiological factors, one of the major risk factors for HCC is a chronic infection of hepatitis B virus (HBV). HBV X protein (HBx) has been identified to play an important role in the HBV-induced HCC pathogenesis since it may interfere with several key regulators of many cellular processes. HBx localization within the cells may be beneficial to HBx multiple functions at different phases of HBV infection and associated hepatocarcinogenesis. HBx as a regulatory protein modulates cellular transcription, molecular signal transduction, cell cycle, apoptosis, autophagy, protein degradation pathways, and host genetic stability via interaction with various factors, including its association with various non-coding RNAs. A better understanding on the regulatory mechanism of HBx on various characteristics of HCC would provide an overall picture of HBV-associated HCC. This article addresses recent data on HBx role in the HBV-associated hepatocarcinogenesis.

Effects of neddylation on viral infection: an overview.

Neddylation is a post-translational modification that plays an important role not only in cancer development but also in regulating viral infection and replication. Upregulation of neddylation occurs in viral infections, and inhibition of neddylation can suppress viral replication. Neddylation is thought to enhance viral protein stability and replication. Neddylation has been reported to enhance the stability of the regulatory hepatitis B virus (HBV) X protein, modulate viral replication, and enhance hepatocarcinogenesis. Inhibition of neddylation using the NEDD8-activating enzyme E1 inhibitor MLN4924 inhibits viral replication, including that of HBV. Understanding of the role of neddylation in viral infections is critical for developing new therapeutic targets and potential treatment strategies. In this review, we discuss recent progress in the understanding of the effects of neddylation during viral infection, particularly in HBV infection, and strategies for curing viral infection by targeting the neddylation pathway.

HBx Modulates Drug Resistance of Sorafenib-Resistant Hepatocellular Carcinoma Cells.

BACKGROUND: Approximately 50% of hepatocellular carcinoma (HCC) arises due to the infection by hepatitis B virus X protein (HBx). Sorafenib, a unique targeted oral kinase inhibitor, is the therapeutic agent of choice for advanced HCC. The mechanism of HBx in drug resistance of sorafenib-resistant HCC cells was evaluated in this study. METHODS: Employing a stepwise increase of the sorafenib content, Hep3B and HepG2 cells were iteratively induced to establish drug-resistant cell lines (Hep3B/R and HepG2/R). The survival rate of Hep3B, Hep3B/R, HepG2, and HepG2/R cells was estimated using the cell counting kit-8 (CCK-8) assay. The IC50 values of sorafenib were calculated, exploring its effects under varying concentrations. The HBx content was quantified via quantitative reverse transcription PCR (RT-qPCR) and Western Blot. HBx overexpression and interfering virus vectors were constructed and transfected into Hep3B/R and HepG2/R cells. Cell viability and metastasis were assessed by colony formation, wound healing, and transwell assays. E-cadherin, N-cadherin, Vimentin, Slug, and Snail content was evaluated via Western Blot. RESULTS: HBx content was significantly elevated in Hep3B/R and HepG2/R subgroups compared to Hep3B and HepG2 subgroups. The proliferation, clonogenicity, invasiveness, and migratory abilities of Hep3B/R and HepG2/R cells in the HBx subgroup were markedly enhanced; E-cadherin content was significantly reduced, whereas the content of N-cadherin, Vimentin, Slug, and Snail was significantly elevated in the HBx subgroup. Conversely, in the sh-HBx subgroup, the proliferation, clonogenicity, invasion, and migration of Hep3B/R and HepG2/R cells were significantly reduced, E-cadherin content was markedly increased, and N-cadherin, Vimentin, Slug, and Snail content was significantly reduced, compared to the sh-negative control (NC) subgroup. CONCLUSIONS: HBx knockout may affect the development of HCC by reducing the proliferation, invasion, and migration of Hep3B/R and HepG2/R cells through the inhibition of Epithelial-Mesenchymal Transition (EMT).

HBx induced upregulation of FATP2 promotes the development of hepatic lipid accumulation.

The hepatitis B Virus X (HBx) protein plays a crucial role in the HBV-induced hepatic steatosis. Fatty acid transport protein 2 (FATP2) is a key protein that is involved in hepatic lipogenesis, and it was found to be highly expressed in various metabolic diseases. However, Whether FATP2 is a key factor in the pathogenesis of HBx-induced hepatic steatosis remains unclear. In this study, we found that FATP2 was up-regulated by HBx in vitro and in vivo and participated in HBx-induced hepatic lipid accumulation. Treatment of HBx-expressing cell lines and mice with FATP2 inhibitor (FATP2i) lipofermata ameliorated HBx-induced lipid accumulation and reduced oxidative stress and inflammation caused by lipid accumulation. Moreover, the liver injury of mouse was restored after FATP2i treatment. In summary, our results reveal that FATP2 is a key driver factor for HBx-induced hepatic lipid accumulation, and inhibition of FATP2 can ameliorates lipid accumulation caused by HBx. This study provides new insights into the mechanism of HBV-induced hepatic steatosis.

Pre- and Post-Transcriptional Control of HBV Gene Expression: The Road Traveled towards the New Paradigm of HBx, Its Isoforms, and Their Diverse Functions.

Hepatitis B virus (HBV) is an enveloped DNA human virus belonging to the Hepadnaviridae family. Perhaps its main distinguishable characteristic is the replication of its genome through a reverse transcription process. The HBV circular genome encodes only four overlapping reading frames, encoding for the main canonical proteins named core, P, surface, and X (or HBx protein). However, pre- and post-transcriptional gene regulation diversifies the full HBV proteome into diverse isoform proteins. In line with this, hepatitis B virus X protein (HBx) is a viral multifunctional and regulatory protein of 16.5 kDa, whose canonical reading frame presents two phylogenetically conserved internal in-frame translational initiation codons, and which results as well in the expression of two divergent N-terminal smaller isoforms of 8.6 and 5.8 kDa, during translation. The canonical HBx, as well as the smaller isoform proteins, displays different roles during viral replication and subcellular localizations. In this article, we reviewed the different mechanisms of pre- and post-transcriptional regulation of protein expression that take place during viral replication. We also investigated all the past and recent evidence about HBV HBx gene regulation and its divergent N-terminal isoform proteins. Evidence has been collected for over 30 years. The accumulated evidence simply strengthens the concept of a new paradigm of the canonical HBx, and its smaller divergent N-terminal isoform proteins, not only during viral replication, but also throughout cell pathogenesis.

Hepatitis B Virus X Protein Modulates p90 Ribosomal S6 Kinase 2 by ERK to Promote Growth of Hepatoma Cells.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), one of the most prevalent malignant tumors worldwide that poses a significant threat to human health. The multifunctional regulator known as Hepatitis B virus X-protein (HBx) interacts with host factors, modulating gene transcription and signaling pathways and contributing to hepatocellular carcinogenesis. The p90 ribosomal S6 kinase 2 (RSK2) is a member of the 90 kDa ribosomal S6 kinase family involved in various intracellular processes and cancer pathogenesis. At present, the role and mechanism of RSK2 in the development of HBx-induced HCC are not yet clear. In this study, we found that HBx upregulates the expression of RSK2 in HBV-HCC tissues, HepG2, and SMMC-7721 cells. We further observed that reducing the expression of RSK2 inhibited HCC cell proliferation. In HCC cell lines with stable HBx expression, RSK2 knockdown impaired the ability of HBx to promote cell proliferation. The extracellularly regulated protein kinases (ERK) 1/2 signaling pathway, rather than the p38 signaling pathway, mediated HBx-induced upregulation of RSK2 expression. Additionally, RSK2 and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were highly expressed and positively correlated in HBV-HCC tissues and associated with tumor size. This study showed that HBx upregulates the expression of RSK2 and CREB by activating the ERK1/2 signaling pathway, promoting the proliferation of HCC cells. Furthermore, we identified RSK2 and CREB as potential prognostic markers for HCC patients.

Hepatitis B virus X protein induces ALDH2 ubiquitin-dependent degradation to enhance alcoholic steatohepatitis.

Background: Excessive alcohol intake with hepatitis B virus (HBV) infection accelerates chronic liver disease progression and patients with HBV infection are more susceptible to alcohol-induced liver disease. Hepatitis B virus X protein (HBx) plays a crucial role in disease pathogenesis, while its specific role in alcoholic liver disease (ALD) progression has not yet been elucidated. Here, we studied the role of HBx on the development of ALD. Methods: HBx-transgenic (HBx-Tg) mice and their wild-type littermates were exposed to chronic plus binge alcohol feeding. Primary hepatocytes, cell lines, and human samples were used to investigate the interaction between HBx and acetaldehyde dehydrogenase 2 (ALDH2). Lipid profiles in mouse livers and cells were assessed by using liquid chromatography-mass spectrometry. Results: We identified that HBx significantly aggravated alcohol-induced steatohepatitis, oxidative stress, and lipid peroxidation in mice. In addition, HBx induced worse lipid profiles with high lysophospholipids generation in alcoholic steatohepatitis, as shown by using lipidomic analysis. Importantly, serum and liver acetaldehyde were markedly higher in alcohol-fed HBx-Tg mice. Acetaldehyde induced lysophospholipids generation through oxidative stress in hepatocytes. Mechanistically, HBx directly bound to mitochondrial ALDH2 to induce its ubiquitin-proteasome degradation, resulting in acetaldehyde accumulation. More importantly, we also identified that patients with HBV infection reduced ALDH2 protein levels in the liver. Conclusions: Our study demonstrated that HBx-induced ubiquitin-dependent degradation of mitochondrial ALDH2 aggravates alcoholic steatohepatitis.

C-Terminal Truncated HBx Facilitates Oncogenesis by Modulating Cell Cycle and Glucose Metabolism in FXR-Deficient Hepatocellular Carcinoma.

Farnesoid X receptor (FXR) is a nuclear receptor known to play protective roles in anti-hepatocarcinogenesis and regulation of the basal metabolism of glucose, lipids, and bile acids. FXR expression is low or absent in HBV-associated hepatocarcinogenesis. Full-length HBx and HBx C-terminal truncation are frequently found in clinical HCC samples and play distinct roles in hepatocarcinogenesis by interacting with FXR or FXR signaling. However, the impact of C-terminal truncated HBx on the progression of hepatocarcinogenesis in the absence of FXR is unclear. In this study, we found that one known FXR binding protein, a C-terminal truncated X protein (HBx C40) enhanced obviously and promoted tumor cell proliferation and migration by altering cell cycle distribution and inducing apoptosis in the absence of FXR. HBx C40 enhanced the growth of FXR-deficient tumors in vivo. In addition, RNA-sequencing analysis showed that HBx C40 overexpression could affect energy metabolism. Overexpressed HSPB8 aggravated the metabolic reprogramming induced by down-regulating glucose metabolism-associated hexokinase 2 genes in HBx C40-induced hepatocarcinogenesis. Overall, our study suggests that C-terminal truncated HBx C40 synergizes with FXR deficiency by altering cell cycle distribution as well as disturbing glucose metabolism to promote HCC development.

Role of AHR, NF-kB and CYP1A1 crosstalk with the X protein of Hepatitis B virus in hepatocellular carcinoma cells.

In this study, it was aimed to elucidate the interaction between aryl hydrocarbon receptor (AHR), nuclear factor-kappa B (NF-kB), and cytochrome P4501A1 (CYP1A1) with hepatitis B virus X protein (HBX) in a human liver cancer cell line (HepG2) transfected with HBX. First, AHR, NF-kB, and CYP1A1 genes were cloned into the appropriate region of the CheckMate mammalian two-hybrid recipient plasmids using a flexi vector system. Renilla and firefly luciferases were quantified using the dual-luciferase reporter assay system to measure the interactions. Secondly, transient transfections of CYP1A1 and NF-kB (RelA) were performed into HBX-positive and HBX-negative HepG2 cells. The mRNA expression of CYP1A1 and NF-kB genes were confirmed with RT-PCR, and cell viability was measured by WST-1. Further verification was assessed by measuring the activity and protein level of CYP1A1. Additionally, CYP1A1/HBX protein-protein interactions were performed with co-immunoprecipitation, which demonstrated no interaction. These results have clearly shown that the NF-kB and AHR genes interact with HBX without involving CYP1A1 and HBX protein-protein interactions. The present study confirms that AHR and NF-kB interaction plays a role in the HBV mechanism mediated via HBX and coordinating the carcinogenic or inflammatory responses; still, the CYP1A1 gene has no effect on this interaction.

Mechanism of cytotoxic T lymphocyte－derived exosomes inhibiting hepatic stellate cell activation / 临床肝胆病杂志

ObjectiveTo investigate whether cytotoxic T lymphocyte （CTL）－derived exosomes can downregulate HBx expression and inhibit hepatic stellate cell （HSC） activation. MethodsThe supernatants of HepG2， HepGA14， and CTL cells were collected to extract exosomes， which were referred to as NC－exo， HBV－exo， and CTL－exo， respectively）. Transmission electron microscopy was used to observe their morphology， and Western Blot was used to measure the expression of the markers of exosomes CD63 and TSG101. NC－exo， HBV－exo， and CTL－exo labeled by BODIPY dye were mixed with HBV－exo at different ratios and were then co－cultured with HSC LX－2 （HSC－LX2）. A fluorescence microscope was used to observe whether exosomes could enter LX－2 cells， and an fluorescence microscope was used to observe cell morphological changes； quantitative real－time PCR （qPCR） was used to measure the expression of the activated biomarkers such as transforming growth factor－β1 （TGF－β1）， ɑ－smooth muscle actin （ɑ－SMA）， and collagen type I （Collagen I） in LX－2 cells. CTL－exo was added to the HepGA14 culture system； then qPCR was used to measure the mRNA expression level of HBV DNA， cccDNA， and HBx in exosomes in HepGA14 cells， and Western Blot was used to measure the protein expression level of HBx in exosomes. The t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsThe exosomes were all microcysts with a double－layer membrane structure and were circular or elliptical in shape， with the expression of the signature proteins CD63 and TSG101， and the vesicles had a diameter of 50－100 nm. The fluorescence microscope showed that exosomes could enter LX－2 cells， and HSC were enlarged with extended cell processes. The results of qPCR showed that there were significant differences in the expression levels of TGF－β1， ɑ－SMA， and Collagen I genes between the NC－exo， HBV－exo， NC－exo+HBV－exo， and Con groups （F=444.678， 417.144， and 571.508， all P<0.05）. After the intervention of HepGA14 cells with CTL－exo， qPCR results showed that compared with the control group， there were significant reductions in the expression levels of HBV DNA and cccDNA in HepGA14 cells （all P<0.05）， the relative mRNA expression level of HBx in exosomes （P<0.05）， and the protein expression level of HBx （P<0.05）. CTL－exo and HBV－exo were mixed at different ratios （2∶1， 5∶1， 10∶1） and were then used for the intervention of LX－2 cells， and qPCR results showed that the expression levels of TGF－β1， ɑ－SMA， and Collagen I genes in LX－2 cells gradually decreased with the increase in the ratio of CTL－exo between groups （P<0.05）. ConclusionCTL－exo can downregulate the protein expression of HBx in HBV－exo to inhibit HSC activation， suggesting that CTL－exo has an anti－hepatitis B liver fibrosis effect.

Aerobic glycolysis enhances HBx-initiated hepatocellular carcinogenesis via NF-κBp65/HK2 signalling.

BACKGROUND: Aerobic glycolysis has been recognized as one of the growth-promoting metabolic alterations of cancer cells. Emerging evidence indicates that nuclear factor κB (NF-κB) plays significant roles in metabolic adaptation in normal cells and cancer cells. However, whether and how NF-κB regulates metabolic reprogramming in hepatocellular carcinoma (HCC), specifically hepatitis B virus X protein (HBx)-initiated HCC, has not been determined. METHODS: A dataset of the HCC cohort from the TCGA database was used to analyse the expression of NF-κB family members. Expression of NF-κBp65 and phosphorylation of NF-κBp65 (p-p65) were detected in liver tissues from HBV-related HCC patients and normal controls. A newly established HBx+/+/NF-κBp65f/f and HBx+/+/NF-κBp65Δhepa spontaneous HCC mouse model was used to investigate the effects of NF-κBp65 on HBx-initiated hepatocarcinogenesis. Whether and how NF-κBp65 is involved in aerobic glycolysis induced by HBx in hepatocellular carcinogenesis were analysed in vitro and in vivo. RESULTS: NF-κBp65 was upregulated in HBV-related HCC, and HBx induced NF-κBp65 upregulation and phosphorylation in vivo and in vitro. Hepatocyte-specific NF-κBp65 deficiency remarkably decreased HBx-initiated spontaneous HCC incidence in HBx-TG mice. Mechanistically, HBx induced aerobic glycolysis by activating NF-κBp65/hexokinase 2 (HK2) signalling in spontaneous hepatocarcinogenesis, and overproduced lactate significantly promoted HCC cell pernicious proliferation via the PI3K (phosphatidylinositide 3-kinase)/Akt pathway in hepatocarcinogenesis. CONCLUSION: The data elucidate that NF-κBp65 plays a pivotal role in HBx-initiated spontaneous HCC, which depends on hyperactive NF-κBp65/HK2-mediated aerobic glycolysis to activate PI3K/Akt signalling. Thus, phosphorylation of NF-κBp65 will be a potential therapeutic target for HBV-related HCC.

Hepatocyte-Derived Prostaglandin E2-Modulated Macrophage M1-Type Polarization via mTOR-NPC1 Axis-Regulated Cholesterol Transport from Lysosomes to the Endoplasmic Reticulum in Hepatitis B Virus x Protein-Related Nonalcoholic Steatohepatitis.

Lipid metabolic dysregulation and liver inflammation have been reported to be associated with nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unclear. Hepatitis B virus x protein (HBx) is a risk factor for NASH. Based on metabolomic and transcriptomic screens and public database analysis, we found that HBx-expressing hepatocyte-derived prostaglandin E2 (PGE2) induced macrophage polarization imbalance via prostaglandin E2 receptor 4 (EP4) through in vitro, ex vivo, and in vivo models. Here, we revealed that the M1-type polarization of macrophages induced by endoplasmic reticulum oxidoreductase-1-like protein α (ERO1α)-dependent endoplasmic reticulum stress was associated with the HBx-related hepatic NASH phenotype. Mechanistically, HBx promoted Niemann-Pick type C1 (NPC1)/oxysterol-binding protein-related protein 5 (ORP5)-mediated cholesterol transport from the lysosome to the endoplasmic reticulum via mammalian target of rapamycin (mTOR) activation. This study provides a novel basis for screening potential biomarkers in the macrophage mTOR-cholesterol homeostasis-polarization regulatory signaling pathway and evaluating targeted interventions for HBx-associated NASH.

Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen.

Background & Aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants. Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression. Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment. Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B. Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.

Long noncoding RNAs in hepatitis B virus replication and oncogenesis.

Several diverse long noncoding RNAs (lncRNAs) have been identified to be involved in hepatitis B virus (HBV) replication and oncogenesis, especially those dysregulated in HBV-related hepatocellular carcinoma (HCC). Most of these dysregulated lncRNAs are modulated by the HBV X protein. The regulatory mechanisms of some lncRNAs in HBV replication and oncogenesis have been characterized. Genetic polymorphisms of several lncRNAs affecting HBV replication or oncogenesis have also been studied. The prognosis of HCC remains poor. It is important to identify novel tumor markers for early diagnosis and find more therapeutic targets for effective treatments of HCC. Some dysregulated lncRNAs in HBV-related HCC may become biomarkers for early diagnosis and/or the therapeutic targets of HCC. This mini-review summarizes these findings briefly, focusing on recent developments.

Hepatitis B Virus Induces Microtubule Stabilization to Promote Productive Infection through Upregulating Microtubule-associated Protein 1S.

Background and Aims: Continuous release and transmission of hepatitis B virus (HBV) is one of the main factors leading to chronic hepatitis B (CHB) infection. However, the mechanism of HBV-host interaction for optimal viral transport is unclear. Hence, we aimed to explore how HBV manipulates microtubule-associated protein 1S (MAP1S) and microtubule (MT) to facilitate its transport and release. Methods: The expression of MAP1S or acetylated MT was investigated by immunofluorescence, RT-PCR, immunoblotting, and plasmid transfection. MAP1S overexpression or knockdown was performed by lentiviral infection or sh-RNA transfection, respectively. HBV DNA was quantified using q-PCR. Results: Significantly higher level of MAP1S in HepG2215 cells compared with HepG2 cells was detected using RT-PCR (p<0.01) and immunoblotting (p<0.001). Notably, stronger MAP1S expression was observed in the liver tissues of patients with CHB than in healthy controls. MAP1S overexpression or knockdown demonstrated that MAP1S promoted MT acetylation and reduced the ratio of HBV DNA copies inside to outside cells. Further, transfection with the hepatitis B virus X protein (HBx)-expressing plasmids induced significantly higher level of MAP1S than that in controls (p<0.0001), whereas HBVX- mutant-encoding HBV proteins (surface antigen, core protein, and viral DNA polymerase) hardly affected its expression. Conclusions: These results demonstrate that HBx induces the formation of stable MTs to promote the release of HBV particles through upregulating MAP1S. Thus, our studies delineate a unique molecular pathway through which HBV manipulates the cytoskeleton to facilitate its own transportation, and indicate the possibility of targeting MAP1S pathway for treatment of patients with CHB.

Hepatitis B virus X protein and hepatitis C virus core protein cooperate to repress E-cadherin expression via DNA methylation.

Dual infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) is closely associated with an increased risk of hepatocellular carcinoma; however, the underlying mechanism is poorly understood. In the present study, we found that HBV X protein (HBx) and HCV core protein work together to inhibit E-cadherin expression in human hepatoma cells. For this effect, they additively increased both the level and activity of enzymes, DNA methyltransferase 1, 3a, and 3b to induce promoter hypermethylation of E-cadherin in a p53-dependent fashion. Their additive effect on E-cadherin levels was reproduced in an in vitro HBV/HCV dual infection system using Huh7D-NTCP cells. As a result, HBV and HCV additively upregulated mesenchymal marker such as N-cadherin, Snail, Twist and Vimentin but cooperatively downregulated epithelial markers such as E-cadherin, Slug and Plakoglobin. In addition, the coinfected cells exhibited faster cell migration and higher invasion ability, as compared with monoinfection, which are hallmarks of epithelial-mesenchymal transition required for the initiation of metastasis in cancer progression.

DNA methyltransferase 1 knockdown reverses PTEN and VDR by mediating demethylation of promoter and protects against renal injuries in hepatitis B virus-associated glomerulonephritis.

BACKGROUND: Aberrant DNA methylation patterns, including hypermethylation of key genes that inhibit fibrosis and inflammation, have been described in human kidney diseases. However, the role of DNA methyltransferase 1 (DNMT1) in hepatitis B virus-associated glomerulonephritis (HBV-GN) remains unclear. METHODS: We explored the underlying mechanism by establishing HBV X protein (HBx) overexpressing renal tubular epithelial (HK-2) cells and human podocytes with DNMT1 knockdown. Using RNA-sequencing to determine the downstream targets of DNMT1 and evaluate its levels of promoter methylation. HBV transgenic mice were used to examine the effects of DNMT1 inhibitor on renal in vivo. RESULTS: DNMT1 was significantly upregulated in the renal tissue of HBV-GN patients, accompanied by injuries of HK-2 cells and podocytes. HBx markedly upregulated DNMT1 and induced epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells and human podocytes. This increased DNMT1 expression was attenuated after DNMT1 knockdown, accompanied by restored HK-2 cells and podocyte injuries resulting from the activation of PI3K/Akt/mTOR and nuclear factor-kappa B (NF-κB) pathways. Hypermethylation of the phosphatase and tensin homolog (PTEN) promoter and vitamin D receptor (VDR) was induced in HBx-overexpressing HK-2 cells and podocytes, respectively, whereas DNMT1 knockdown effectively corrected these alterations. Furthermore, PTEN and VDR ablation resulted in marked EMT and inflammation induction in HBx-overexpressing HK-2 cells and human podocytes even with DNMT1 knockdown. Downregulation of the PI3K/Akt/mTOR-related pathway attenuated HBx-induced EMT and inflammation in HK-2 cells. Luciferase reporter assay revealed VDR as a direct target of the Snail family transcriptional repressor 1 (SNAI1) in HBx-overexpressing podocytes. DNA methylation inhibitor 5-azacytidine alleviated urinary protein and renal inflammation in HBV transgenic mice via PTEN-PI3K/Akt signaling and VDR signaling axis. CONCLUSIONS: Our study clarifies the potential epigenetic mechanisms underlying HBx-induced renal injuries in HBV-GN and the renoprotective effects of inhibiting DNMT1, which can provide important insights into the development of treatments for HBV-GN.

Inhibition of Protease Activated Receptor 2 Attenuates HBx-Induced Inflammation and Mitochondria Oxidative Stress.

Background: Hepatitis B virus (HBV) infection is one of the global public problems. Among the known infection cases, HBV X protein (HBx) is one of the key inducements of viral replication and host infection. This study was aimed to uncover the role of protease activated receptor 2 (PAR2) on HBx-induced liver injury. Methods: A PAR2-KO mouse model expressing HBx was constructed using hydrodynamics-based in vivo gene transfection method. In addition, pcDNA3.1-HBx was used to over-express HBx in LO2 cells. The effects of HBx overexpression on inflammation and mitochondria oxidative stress were evaluated. Results: We found that PAR2 protein level was increased by HBx overexpression. The enforced HBx inhibited LO2 cells apoptosis. Meanwhile, HBx induced inflammation reactions through promoting the secretion of pro-inflammatory cytokines such as TNF-α, IL-6, and CXCL-2. Overexpressed HBx also resulted in mitochondria oxidative stress by upregulation of ROS level and downregulation of MMP and ATP. However, in FSLLRY-NH2 (PAR2 antagonist) treated LO2 cells or PAR2-KO mice, PAR2 blockade reversed the above adverse effects of HBx on liver cells or tissues. Conclusion: Inhibition of PAR2 may suppress inflammation and mitochondria oxidative stress caused by HBx, pointing out the potential application values of PAR2 antagonist on the treatment of HBV infection in clinic.

Novel X gene point mutations in chronic hepatitis B and HBV related cirrhotic patients.

INTRODUCTION: HBx is a multifunctional modulator viral protein with key roles in various biological processes such as signal transduction, transcription, proliferation, and cell apoptosis. Also, HBx has an important role in the progression of cirrhosis and hepatocellular carcinoma (HCC). This study aimed to determine mutations in X gene, enhancer II (EnhII), and basal core promoter (BCP) of genotype D of Hepatitis B Virus (HBV) in cirrhotic and chronic HBV patients. MATERIAL AND METHODS: This cross-sectional study was performed on 68 cases with chronic HBV (cHBV) and 50 cases with HBV related cirrhosis. Serum samples were obtained for genomic DNA extraction. Semi-nested PCR was used to amplify the HBx region. Point mutations in the HBx region were detected by sequencing. RESULT: Novel mutations were detected, including C1491G, C1500T, G1613T, and G1658T in the N-terminal of the X gene. The frequency of C1481T/G1479A, T1498C, C1500T, G1512A, A1635T, C1678T, A1727T, and A1762T/ G1764A/ C1773T was significantly higher in cirrhotic patients compared to chronically HBV infected ones. A higher rate of A1635T, C1678T, A1727T, A1762T, G1764A, and C1773T was observed in cirrhotic patients. CONCLUSION: Our findings showed that the frequency of mutations in the basal-core promoter, enhancer II, and regulatory region of the HBx gene was more seen in cirrhotic patients than in chronic HBV cases. Novel mutations were detected in the HBx gene, causing amino acid substitutions; however, the clinical impact of these novel mutations is yet to be cleared.

Ubiquitin-specific peptidase 22 (USP22) regulates the level and function of hepatitis B virus X protein / 中华微生物学和免疫学杂志

Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.