Protein glycosylation plays an important role in the pathogenesis and progression of various liver diseases. However, little is known about the precise alterations in protein glycosylation or the potential correlation between glycan-related genes expression and glycan profiles in liver fibrosis. The aim of the study was to investigate potential associations between glycan-related genes expression and glycan profiles to evaluate liver fibrosis in a mouse model. Analyses of glycan-related genes expression and glycan profiles were performed using oligonucleotide microarrays and lectin microarrays, respectively. Real-time PCR and Western blot were used to confirm any altered glycan-related genes expression levels and protein levels. Moreover, altered glycan patterns on the surface of hepatocytes were verified by lectin histochemistry. These results revealed that the mRNA levels of 10 glycan-related genes were significantly altered in fibrotic liver. Furthermore, we observed an increase in multivalent sialic acid, poly-LacNAc, sialyl-T-antigen, Fucoseα-1,3/6GlcNAc, and GalNAcα1–3Gal in fibrotic liver specimens, whereas GlcNAc oligomers was decreased in fibrotic liver. Our findings indicated that the synthetic pathway of “Tn antigen → T antigen (core-1) → sialyl-T antigen” was activated for O-glycan during the process of liver fibrosis.Keywords: gene expression microarray; glycan structure; glycan-related genes; hepatic fibrosis; lectin histochemistry; lectin microarray; tumorigenesis;

Cellular plasticity has an important role in the progression of hepatocellular carcinoma (HCC). In this study, the involvement of a TGF-β1-CD147 self-sustaining network in the regulation of the dedifferentiation progress was fully explored in HCC cell lines, hepatocyte-specific basigin/CD147-knockout mice and human HCC tissues. We demonstrated that TGF-β1 stimulation upregulated CD147 expression and mediated the dedifferentiation of HCC cells, whereas all-trans-retinoic acid induced the downregulation of CD147 and promoted differentiation in HCC cells. Overexpression of CD147 induced the dedifferentiation and enhanced the malignancy of HCC cells, and increased the transcriptional expression of TGF-β1 by activating β-catenin. CD147-induced matrix metalloproteinase (MMP) production activated pro-TGF-β1. The activated TGF-β1 signaling subsequently repressed the HNF4α expression via Smad-Snail1 signaling and enhanced the dedifferentiation progress. Hepatocyte-specific basigin/CD147-knockout mice decreased the susceptibility to N-nitrosodiethylamine-induced tumorigenesis by suppressing TGF-β1-CD147 signaling and inhibiting dedifferentiation in hepatocytes during tumor progression. CD147 was positively correlated with TGF-β1 and negatively correlated with HNF4α in human HCC tissues. Positive CD147 staining and lower HNF4α levels in tumor tissues were significantly associated with poor survival of patients with HCC. The overexpression of HNF4α and Smad7 and the deletion of CD147 by lentiviral vectors jointly reprogrammed the expression profile of hepatocyte markers and attenuated malignant properties including proliferation, cell survival and tumor growth of HCC cells. Our results highlight the important role of the TGF-β1-CD147 self-sustaining network in driving HCC development by regulating differentiation plasticity, which provides a strong basis for further investigations of the differentiation therapy of HCC targeting TGF-β1 and CD147.

Oncolytic virus which arms the therapeutic gene to enhance anti-tumor activity is a prevalent strategy to improve oncovirotherapy of cancer. Newcastle disease virus (NDV) is a naturally oncolytic virus used for cancer therapy. Previously, we generated a mouse-human chimeric HAb18 antibody (cHAb18) against tumor-associated antigen CD147 and demonstrated the inhibition of invasion and migration of hepatocellular carcinoma (HCC) cells. Here, we constructed a recombinant NDV carrying intact cHAb18 gene (rNDV-18HL) based on Italien strain using a reverse genetics system.Recombinant rNDV-18HL was generated using reverse genetics technology. The characteristics of virally expressed cHAb18 antibody were identified by western blot, enzyme-linked immunosorbent assay, transwell invasion assay, and surface plasmon resonance technology. The biodistribution of recombinant rNDV-18HL using orthotopic xenograft mouse model was assessed with living imaging and immunohistochemistry. Kaplan-Meier survival curves and the log-rank test were performed to analyze the anti-tumor activity of rNDV-18HL.The cHAb18 was produced in rNDV-18HL-infected cells followed by releasing into the supernatant by cytolysis. The rNDV-18HL-encoded cHAb18 antibody kept affinity for CD147 and showed inhibiting the migration and invasion of HCC cells. Viral replication and virulence were not attenuated by the incorporation of cHAb18 gene which significantly enhanced the suppression of relict tumor cell migration. The rNDV-18HL selectively replicated in orthotopic HCC xenografts leading to cHAb18 expression in situ, which induced the tumor necrosis, reduced the intrahepatic metastasis, and prolonged the survival in mice.This study provides a new strategy of arming oncolytic NDV with therapeutic antibody to enhance anti-tumor efficacy of cancer therapy.

Overexpression of CD147/basigin in hepatic cells promotes the progression of hepatocellular carcinoma (HCC). Whether CD147 also expressed in liver non-parenchymal cells and associated with HCC development was unknown. The aim of the study was to explore time-dependent cell expression patterns of CD147 in a widely accepted N-diethylnitrosamine/phenobarbital (DEN/PB)-induced HCC mouse model. Liver samples collected at month 1–12 of post-DEN/PB administration were assessed the localization of CD147 in hepatocytes, endothelial cells, hepatic stellate cells, and macrophages. Immunohistochemistry analysis showed that CD147 was upregulated in liver tumors during month 1–8 of DEN/PB induction. Expression of CD147 was positively correlated with cytokeratin 18, a hepatocyte marker (r = 0.7857, P = 0.0279), CD31 (r = 0.9048, P = 0.0046), an endothelial cell marker, and CD68, a macrophage marker (r = 0.7619, P = 0.0368). A significant correlation was also observed between CD147 and alpha-smooth muscle actin (r = 0.8857, P = 0.0333) at DEN/PB initiation and early stage of tumor formation. Immunofluorescence and fluorescence in situ hybridization showed that CD147 co-expressed with cytokeratin 18, CD31, alpha-smooth muscle actin, and CD68. Moreover, there existed positive correlations between CD147 and microvessel density (r = 0.7857, P = 0.0279), CD147 and Ki-67 (r = 0.9341, P = 0.0022) in the development of DEN/PB-induced HCC. In conclusion, our results demonstrated that CD147 was upregulated in the liver parenchymal and mesenchymal cells and involved in angiogenesis and tumor cell proliferation in the development of DEN/PB-induced HCC.

CD147 is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, breast, and liver and therefore it is a potentially unique therapeutic target for these diverse tumor types. We previously generated a murine antibody HAb18 which suppresses matrix met al.loproteinase-2 and matrix metalloproteinase-9 secretion, attenuates cell invasion by blocking the CD147 molecule in tumor cells. Here, we generated a chimeric antibody containing the variable heavy and variable light chains of murine HAb18 and the constant regions of human IgG1γ1 and human κ chain as a potential therapeutic agent (designated cHAb18). Quantitative measurement of cHAb18 antibody affinity for antigen CD147 with surface plasmon resonance showed the equilibrium dissociation constant KD was 2.66 × 10−10 mol/L, similar to that of KD 2.73 × 10−10 mol/L for murine HAb18. cHAb18 induced antibody-dependent cell-mediated cytotoxicity in two hepatocellular carcinoma cell lines, SMMC-7721 and Huh-7 cells. It inhibited cancer invasion and migration in hepatocellular carcinoma cells by specifically blocking CD147. Except for the depression of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions, cHAb18 antibody suppressed cell motility by rearrangement of actin cytoskeleton, which was probably induced by decreasing the phosphorylation of focal adhesion kinase, phosphatidylinositide-3 kinase (PI3K), Akt, and Girdin in the integrin signaling pathway. In an orthotopic model of hepatocellular carcinoma in BALB/c nude mice, cHAb18 treatment effectively reduced the tumor metastasis in liver and prolonged the survival. These findings reveal new therapeutic potential for cHAb18 antibody targeting CD147 on tumor therapy.

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-β1 (TGF-β1). The human HSCs, LX-2 were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.Highlights► We predict glycosylation site in proteins which are related to the activation of HSCs. ► The alteration of protein glycosylation in the activated LX-2 is detected and validated. ► Tn and T antigen increase significantly in the activated LX-2. ► Fucose residues with various linkages distribute in different regions of activated LX-2. ► Biantennary N-glycans binder PHA-E showed increased signal on the cell membrane of the activated LX-2.

Epithelial–mesenchymal transition (EMT) induced by transforming growth factor-β (TGF-β) is implicated in hepatocarcinogenesis and hepatocellular carcinoma (HCC) metastasis. HAb18G/CD147, which belongs to the CD147 family, is an HCC-associated antigen that has a crucial role in tumor invasion and metastasis. The goal of this study was to investigate the role of HAb18G/CD147 during EMT in hepatocarcinogenesis. Human normal hepatic cell lines QZG and L02, primary mouse hepatocytes and nude mouse models were used to determine the role of HAb18G/CD147 in EMT, and the involvement of the TGF-β-driven pathway. A dual-luciferase reporter assay and ChIP were used to investigate the transcriptional regulation of the CD147 gene. Samples from patients with liver disease were assessed to determine the relationship between HAb18G/CD147 and typical markers for EMT. Our results show that upregulation of HAb18G/CD147 is induced by TGF-β coupled with downregulation of E-cadherin and upregulation of N-cadherin and vimentin. The expression of HAb18G/CD147 is controlled by the cell survival PI3K/Akt/GSK3β signaling pathway, and is directly regulated by the transcription factor Slug. Transfection of CD147 also induces an elevated expression of TGF-β. CD147-transfected hepatocytes have mesenchymal phenotypes that accelerate tumor formation and tumor metastasis in vivo. Immunohistochemistry analysis shows a negative correlation between HAb18G/CD147 and E-cadherin expression (rs=−0.3622, P=0.0105), and a positive correlation between HAb18G/CD147 and Slug expression (rs=0.3064, P=0.0323) in human HCC tissues. Our study uncovers a novel role of HAb18G/CD147 in mediating EMT in the process of HCC progression and showed that CD147 is a Slug target gene in the signaling cascade TGF-βPI3K/AktGSK3βSnailSlugCD147. Our results suggest that CD147 may be a potential target for the treatment and prevention of HCC.

Newcastle disease virus (NDV) Italien, a velogenic strain, is an oncolytic virus that is considered to be a potential agent for antitumor viral therapy. We constructed three helper plasmids expressing the NP, P and L genes of NDV Italien based on the eukaryotic expression plasmid pcDNA3.1(+). The minigenome consisting of the 3′ leader and 5′ trailer regions of NDV Italien flanking a reporter gene encoding firefly luciferase was constructed to examine the efficacy of the three helper plasmids in viral genome replication and transcription. After co-transfection of BSR-T7/5 cells with the three helper plasmids and the minigenome plasmid, replication of minigenome RNA was evaluated by determining luciferase activity. In the minigenome rescue system, expression of the reporter gene was detected. Our results indicate that the three proteins NP, P, and L are correctly expressed and can assemble into a functional ribonucleoprotein complex that effectively directs the transcription of minigenome RNA.

We determined the complete genome sequence of strain Italien, a virulent and oncolytic strain of Newcastle disease virus (NDV) by direct nucleotide sequencing of RT-PCR products, a size of 15,186 nucleotides (nt). Comparison of six coding genes and non-coding regions of Italien with those of the other 25 sequenced strains revealed NDV Herts/33 was the most similar strain with Italien. The gene encoding the RNA dependent RNA polymerase was the most highly conserved, while the gene encoding phosphoprotein was the most highly variable. The HN and F proteins of Italien have been modeled on the crystal structure in order to study the structural characteristics. Interaction between the HN protein and the heptad repeat B (HRB) region of F protein was analyzed in silico by molecular docking predicted five critical residues I133, V142, D143, R480, and K567 on HN protein. Identification of amino acid residues that could be crucial for this interaction provides working hypotheses for subsequent studies.