Dong Xi

Hepatitis B Virus-induced hFGL2 Transcription Is Dependent on c-Ets-2 and MAPK Signal Pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin plays a pivotal role in the pathogenesis of experimental and human fulminant and chronic viral hepatitis. To define the transcription factor(s) and upstream signal transduction pathways involved in the transcription of human FGL2 (hFGL2) in response to hepatitis B (HB) virus, hepatitis B core (HBc), hepatitis B virus S protein (HBs), or hepatitis B virus X protein (HBx) protein, expression plasmids were cotransfected with an hFGL2 promoter luciferase reporter construct into Chinese hamster ovary and HepG2 cells, respectively. HBc and HBx proteins, but not HBs protein, enhanced hFGL2 transcription in both cell lines. A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hFGL2 gene transcription in response to both HBc and HBx proteins. c-Ets-2 was shown to be translocated to the nucleus in association with hFGL2 expression in response to both HBc and HBx proteins. Short hairpin RNA (shRNA) interference of c-Ets-2 expression inhibited hFGL2 gene transcription by 64.8 and 60.0% in response to HBc and HBx, respectively. c-Ets-2 protein was highly expressed in peripheral blood mononuclear cells from patients with severe chronic hepatitis B (CHB) in contrast to patients with mild CHB. Increased phosphorylation of ERK and JNK was detected in peripheral blood mononuclear cells from patients with severe CHB. ERK inhibitor PD098059 or ERK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBc, whereas JNK inhibitor SP600125 or JNK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBx. In conclusion, HBc and HBx proteins enhance transcription of hFGL2 through c-Ets-2 dependent on MAPK signal pathways.

Downregulation of FGL2/prothrombinase delays HCCLM6 xenograft tumour growth and decreases tumour angiogenesis

Fibrinogen‐like protein 2 (FGL2), which directly generates thrombin from prothrombin without activation of the conventional coagulation cascade, was shown to be overexpressed in various human malignant tumours.

The detection of HBV DNA with gold nanoparticle gene probes

Abstract Objective Gold nanoparticle Hepatitis B virus(HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Methods Alkanethiol modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA gene probes, through covalent binding of Au-S. By using a fluorescence-based method, the number of thiolderivatized, single-stranded oligonucleotides and their hybridization efficiency with complementary oligonucleotides in solution was determined. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotides serving as detection probes, and oligonucleotides im-mobilized on a nylon membrane surface acting as capturing probes, HBV DNA was detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. The modified nanoparticle HBV DNA gene probes were also used to detect the HBV DNA extracted from serum in patients with hepatitis B. Results Compared with bare Au nanoparticles, oligonucleotide modified nanoparticles had a higher stability in NaCl solution or under high temperature environment and the absorbance peak of modified Au nanoparticles shifted from 520 nm to 524 nm. For Au nanoparticles, the maximal oligonucleotide surface coverage of hexaethiol 30-mer oligonucleotide was(132 ± 10) oligonucleotides per nanoparticle, and the percentage of hybridization strands on nanoparticles was(22 ± 3%). Based on a two-probe sandwich hybridization/nanoparticle amplification/silver staining enhancement method, Au nanoparticle gene probes could detect as low as 10−11 mol/L composite HBV DNA molecules on a nylon membrane and the PCR products of HBV DNA visually. As made evident by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules in liquid. Conclusion Our results showed that successfully prepared Au nanoparticle HBV DNA gene probes could be used to detect HBV DNA directly. The detection-visuallized method has many advantages, including high sensitivity, simple operation and low cost. This technique has potential applications in many fields, especially in multi-gene detection chips.

The Nucleocapsid Protein of SARS-CoV Induces Transcription of hfgl2 Prothrombinase Gene Dependent on C/EBP Alpha

Abstract Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.

Virus-induced hepatocellular carcinoma with special emphasis on HBV

Hepatocellular carcinoma (HCC) is a common malignant tumor with high lethality, and the hepatitis B virus (HBV) is a chief cause. HBV can accelerate HCC via multiple mechanisms. First, HBV induces immune reactions that lead to repeated hepatic inflammation, fibrosis and a deficient immune microenvironment. Subsequently, HBV can modify host genes near the insertion point through DNA integration to cause host cell genome instability and to generate carcinogenic fusion proteins. Additionally, HBV expresses diverse active proteins, especially HBx and HBs, which have a range of transactivation functions such as regulation of apoptosis, interference with intracellular signaling pathways, and alteration of epigenetics. Currently, primary prevention measures for HBV-induced HCC focus on vaccination and antiviral treatment. Here, we report the epidemiology, the molecular mechanism and the progress in therapeutic strategies for controlling HBV-induced HCC.

Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.

Adenovirus-mediated artificial microRNA against human fibrinogen like protein 2 inhibits hepatocellular carcinoma growth.

Human fibrinogen‐like protein 2 (hFGL2) is overexpressed in various human solid tumors and is associated with tumor growth, invasion and angiogenesis of hepatocellular carcinoma (HCC) LM6 cells. In the present study, an adenoviral vector expressing an artificial microRNA (miRNA) against hfgl2 (Ad‐hfgl2‐miRNA) was used to evaluate the effect of FGL2 knockdown on cell proliferation and tumor growth of HCCLM6 xenografts.

IL-33 protects murine viral fulminant hepatitis by targeting coagulation hallmark protein FGL2/fibroleukin expression.

HighlightsMHV‐3 infection causes IL‐33 levels increasing and developing of fulminant hepatitis.IL‐33 treatment leads to attenuation of the disease and remarkable reduction of FGL2.In vitro IL‐33 administration prevents FGL2 secretion by macrophages.IL‐33 had marked effects on treating viral fulminant hepatitis by targeting FGL2. Abstract Fulminant hepatitis (FH) is characterized by rapid liver failure and high mortality. The pathogenesis of viral FH includes virus‐induced immune activation, inflammation, and subsequent hepatic apoptosis and necrosis. However, the mechanisms that underlie FH progression are unclear. IL‐33 is a member of the IL‐1‐related cytokines, considered to be an “alarmin” that participates in various diseases, but its precise role in the coagulation of FH is not very clear. In our study, we found that IL‐33 is significantly elevated in mice infected with murine hepatitis virus strain 3 (MHV‐3). This is accompanied by an increase in pro‐coagulant fibrinogen‐like protein 2 (FGL2) in the liver. Previous studies have suggested that an increase in FGL2 is diagnostic of FH and liver necrosis, and animals with no FGL2 had better survivorship during FH. Our studies showed that IL‐33 administration in a MHV‐3 infection promoted survival during FH, with a significant reduction in FGL2 expression and liver inflammation. In vitro IL‐33 treatment abrogated MHV‐3 and IFN‐&ggr; induced FGL2 expression in RAW264.7 and THP‐1 cells, respectively. In conclusion, our research suggests that IL‐33 protects against viral fulminant hepatitis in mice by antagonizing expression of the pro‐coagulant protein FGL2.

CD4-CD8-T cells contribute to the persistence of viral hepatitis by striking a delicate balance in immune modulation.

Abstract Viral hepatitis remains the most common cause of liver disease and a major public health problem. Here, we focus on the role of CD4 CD8 double negative T (DN T) cells involved in the mechanisms of viral persistence in hepatitis. C3H/HeJ mice infected with murine hepatitis virus strain 3 (MHV-3) were used to display chronic viral hepatitis. DN T cells dramatically increased in MHV-3 infected mice. Adoptive transfer of DN T cells from MHV-3 infected mice led to a significant increase in mice survival. The DN T cells with production of IFN-γ and IL-2 are able to kill virus-specific CD8+ T cells via the Fas/FasL dependent pathway. The delicate balance of multiple effects of DN T cells may lead to viral persistence in MHV-3 induced hepatitis. In short, our study identified DN T cells contributing to viral persistence in MHV-3 induced hepatitis in C3H/HeJ mice, which provides a rationale for modulating DN T cells for the management of viral hepatitis.

Construction of mTNFR1shRNA plasmid and its biological effects on MHV-3 induced fulminant hepatitis in BALB/cJ mice.

Previous study on TNFR1-mediated hepatocyte apoptosis has been implicated in the development of fulminant viral hepatitis. To interfere with the potentially effective target, plasmid named p-mTNFR1shRNA complimentary to the sequence responsible for mTNFR1 was also constructed and further confirmed by sequence analysis. To investigate the effect of mTNFR1shRNA plasmid on mTNFR1 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model. By hydrodynamic injection of mTNFR1shRNA plasmid, the survival rate of mice, hepatic pathological change were examined and compared between mice with/without mTNFR1shRNA plasmid intervention. The expression of mTNFR1 was detected by Real-time PCR, immunohistochemistry staining. The mTNFR1shRNA plasmid significantly reduced mTNFR1 expression in vivo, markedly ameliorates inflammatory infiltration, prolonged the survival time period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in inhibiting TNFR1 expression, which has been reported to be involved in the development of a variety of diseases including fulminant viral hepatitis and severe chronic hepatitis B.