Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication

Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes

Lower Expression of MicroRNA-155 Contributes to Dysfunction of Natural Killer Cells in Patients with Chronic Hepatitis B

MicroRNAs have been reported to be regulated in different ways in a variety of liver diseases. As a key modulator of cellular function in both innate and adaptive immunity, the role of miR-155 in chronic hepatitis B virus infection remains largely unknown. Here, we investigated the expression and function of miR-155 in chronic hepatitis B (CHB) patients. It was found that miR-155 expression in peripheral blood mononuclear cells (PBMCs) was lower in CHB patients than healthy controls (HC). Among CHB infection, immune-active (IA) patients with abnormal alanine aminotransferase (ALT) levels had relatively higher miR-155 expression in PBMCs and serum than immune-tolerant carriers, but were comparable to inactive carriers. Moreover, there was a positive correlation between miR-155 expression and ALT levels in CHB patients. Particularly, miR-155 expression in natural killer (NK) cells was significantly downregulated in IA patients compared with HC. Inversely, suppressor of cytokine signaling 1 (SOCS1), a target of miR-155, was upregulated in NK cells of IA patients. Overexpression of miR-155 in NK cells from IA patients led to a decrease in SOCS1 expression and an increase of IFN-γ production. Finally, accompanied by the normalization of ALT, miR-155 expression in PBMCs gradually decreased during telbivudine or peg-IFN-α-2a therapy. Interestingly, higher miR-155 expression at baseline was associated with better response to telbivudine therapy, but not peg-IFN-α-2a. In conclusion, our data suggested that miR-155 downregulation in NK cells of IA patients impaired IFN-γ production by targeting SOCS1, which may contribute to immune dysfunction during CHB infection. Additionally, baseline miR-155 expression could predict the treatment response to telbivudine therapy

Fucoidan from Fucus vesiculosus suppresses hepatitis B virus replication by enhancing extracellular signal-regulated Kinase activation

Abstract Background Hepatitis B virus (HBV) infection is a serious public health problem leading to cirrhosis and hepatocellular carcinoma. As the clinical utility of current therapies is limited, the development of new therapeutic approaches for the prevention and treatment of HBV infection is imperative. Fucoidan is a natural sulfated polysaccharide that extracted from different species of brown seaweed, which was reported to exhibit various bioactivities. However, it remains unclear whether fucoidan influences HBV replication or not. Methods The HBV-infected mouse model was established by hydrodynamic injection of HBV replicative plasmid, and the mice were treated with saline or fucoidan respectively. Besides, we also tested the inhibitory effect of fucoidan against HBV infection in HBV-transfected cell lines. Results The result showed that fucoidan from Fucus vesiculosus decreased serum HBV DNA, HBsAg and HBeAg levels and hepatic HBcAg expression in HBV-infected mice. Moreover, fucoidan treatment also suppressed intracellular HBcAg expression and the secretion of the HBV DNA as well as HBsAg and HBeAg in HBV-expressing cells. Furthermore, we proved that the inhibitory activity by fucoidan was due to the activation of the extracellular signal-regulated kinase (ERK) pathway and the subsequent production of type I interferon. Using specific inhibitor of ERK pathway abrogated the fucoidan-mediated inhibition of HBV replication. Conclusion This study highlights that fucoidan might be served as an alternative therapeutic approach for the treatment of HBV infection

Tripartite Motif-Containing Protein 65 (TRIM65) Inhibits Hepatitis B Virus Transcription

Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65’s antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins’ capacity to inhibit HBV transcription and highlight TRIM65’s pivotal role in this process

Biogenesis and molecular characteristics of serum hepatitis B virus RNA

HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis

Elevated expression of HIGD1A drives hepatocellular carcinoma progression by regulating polyamine metabolism through c-Myc–ODC1 nexus

Abstract Background Hypoxia contributes to cancer progression through various molecular mechanisms and hepatocellular carcinoma (HCC) is one of the most hypoxic malignancies. Hypoxia-inducible gene domain protein-1a (HIGD1A) is typically induced via epigenetic regulation and promotes tumor cell survival during hypoxia. However, the role of HIGD1A in HCC remains unknown. Methods HIGD1A expression was determined in 24 pairs of human HCC samples and para-tumorous tissues. Loss-of-function experiments were conducted both in vivo and in vitro to explore the role of HIGD1A in HCC proliferation and metastasis. Results Increased HIGD1A expression was found in HCC tissues and cell lines, which was induced by hypoxia or low-glucose condition. Moreover, HIGD1A knockdown in HCC cells arrested the cell cycle at the G2/M phase and promoted hypoxia-induced cell apoptosis, resulting in great inhibition of cell proliferation, migration, and invasion, as well as tumor xenograft formation. Interestingly, these anti-tumor effects were not observed in normal hepatocyte cell line L02. Further, HIGD1A knockdown suppressed the expression of ornithine decarboxylase 1 (ODC1), a rate-limiting enzyme of polyamine metabolism under c-Myc regulation. HIGD1A was found to bind with the c-Myc promoter region, and its knockdown decreased the levels of polyamine metabolites. Consistently, the inhibitory effect on HCC phenotype by HIGD1A silencing could be reversed by overexpression of c-Myc or supplementation of polyamines. Conclusions Our results demonstrated that HIGD1A activated c-Myc–ODC1 nexus to regulate polyamine synthesis and to promote HCC survival and malignant phenotype, implying that HIGD1A might represent a novel therapeutic target for HCC

Additional file 1 of HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway

Additional file 1: Table S1. The shRNA sequences of HIGD2A and the primer sequences used in real-time PCR analysis. Figure S1. Diagnostic and prognostic value of HIGD2A expression and its correlation with clinical features. A Representative immunohistochemistry images of HIGD2A expression in normal liver tissues and liver cancer tissues from the HPA. B The protein level of HIGD2A in normal hepatocyte L02 cell line and different liver cancer cell lines. C–F HIGD2A expression in different status of T stage (C), pathologic stage (D), vascular invasion (E) and OS event (F). G–J Kaplan–Meier plots of OS for HIGD2A expression levels in subgroups including T stage: T1 (G), N stage: N0 (H), M stage: M0 (I) and pathologic stage: stage I (J). K Nomogram for OS prediction, with T stage, N stage, M stage, histologic grade and expression of HIGD2A applied as parameters. L Calibration curves of the nomogram for 1-, 3-, 5-year survival prediction. *P < 0.05, **P < 0.01. Figure S2. Knockdown of HIGD2A impedes MHCC97H cells proliferation and migration. A Western blot assay for total HIGD2A protein expression in HIGD2A-knockdown MHCC97H cells. B Left, colony formation of MHCC97H cells transfected shCtrl or shHIGD2A.1. Right, quantification of colony formation based on three independent assays. C The proliferation ability of MHCC97H cells infected with shCtrl lentivirus or shHIGD2A.1 lentivirus was measured by CCK8 assay at the indicated time points. D, E Flow cytometry analysis of Annexin V/7-AAD double stained HepG2, Huh7 and MHCC97H cells transfected shCtrl or shHIGD2A.1. Representative flow cytometric plot (D) and the proportion of apoptosis cells (E). F Annexin V/7-AAD apoptosis assay of L02 cells after HIGD2A gene silencing. Left, representative flow cytometric plot. Right, the proportion of apoptosis cells. G Left, cell migration ability of MHCC97H cells transfected shCtrl or shHIGD2A.1 was analyzed by Transwell assay. Right, quantitative data of migrated cells. Results shown are mean ± SEM. an unpaired t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Scale bar, 100 µm. Figure S3. HIGD2A knockdown inhibited the proliferation and migration of HCC cells in vitro. A, B Colony formation experiments for the effect of HIGD2A knockdown with shRNA on the proliferation of HepG2 and Huh7 cells. C, D The effect of HIGD2A knockdown on the growth of HepG2 and Huh7 cells was detected by CCK8 assays. E The effect of HIGD2A knockdown on cell apoptosis was detected by western blot. F, G Transwell chamber was used to evaluated the effect of HIGD2A knockdown on the migration of HepG2 and Huh7 cells. Results shown are mean ± SEM. an unpaired t-test was used. *P < 0.05, **P < 0.01 and ***P < 0.001. Scale bar, 100 µm. Figure S4. Depletion of HIGD2A induces mitochondrial stress in MHCC97H. A, B Intercellular ATP level in MHCC97H (A) and L02 (B) cells transfected with shCtrl or shHIGD2A.1. C Immunofluorescent (IF) images showing mitochondria morphology in MHCC97H transfected with shCtrl or shHIGD2A.1. Left, representative IF images (magenta, MitoTracker; yellow, DAPI) and 3D mitochondria reconstruction of shCtrl- and shHIGD2A-infected MHCC97H cell. Scale bar, 5 μm; Sphericity heat map, 0.326–0.915. Right, sphericity analysis of 3D reconstructed mitochondria (results are presented as mean ± SD). D Left, oxygen consumption rate (OCR) in MHCC97H transfected with shCtrl or shHIGD2A.1 was measured by seahorse analyzer. Right, basal respiration rate and maximal respiration capacity are shown. E Comparison of Basal Respiration among L02 and HCC cells. Results shown are mean ± SEM. A Mann–Whitney test was used. *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant

Additional file 1 of Elevated expression of HIGD1A drives hepatocellular carcinoma progression by regulating polyamine metabolism through c-Myc–ODC1 nexus

Supplementary Material 1