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Liou L.-B.,Chang Gung Memorial Hospital at Lin kou | Liou L.-B.,Chang Gung University | Huang C.-C.,Chang Gung Memorial Hospital at Lin kou
PLoS ONE | Year: 2016

Objective: We attempted to determine whether the level of enzymes sialyltransferase (ST) and neuraminidase (Neu) and sialic acid (SIA) in patients with systemic lupus erythematosus (SLE) correlates with the SLE Disease Activity Index (SLEDAI) and in patients with rheumatoid arthritis (RA) correlates with the Disease Activity Score28 (DAS28). Methods: We examined cell-surface levels of ST6Gal-1, Neu1, ST3Gal-1, Neu3, α-2,6-SIA, and α-2,3-SIA by using fluorescent anti-enzyme antibodies, fluorescent-conjugated Sambucus nigra lectin, and fluorescent-conjugated Maackia amurensis lectin on blood cells in SLE and RA patients and assessed correlations of these levels with SLEDAI and with DAS28. Areas under the curve (AUC) were calculated for different variables against SLEDAI. Results: The B-cell ST3Gal-1/Neu3 ratio positively correlated with SLEDAI scores (ρ = 0.409 and P = 0.002, statistically significant after Bonferroni' correction for multiple analyses.). It was supported by the inverse correlation of B-cell Neu3 levels with SLEDAI scores (ρ = -0.264, P = 0.048). The B-cell ST3Gal-1/Neu3 ratio against SLEDAI yielded an AUC of 0.689, which was comparable to that of anti-dsDNA levels at 0.635. In contrast, both ST3Gal-1 and Neu3 levels of RA B cells (r = 0.376, P = 0.013; r = 0.425, P = 0.005, respectively) correlated positively with high disease-activity DAS28 scores. Conclusion: B-cell ST3Gal-1/Neu3 ratios in SLE and B-cell ST3Gal-1 and Neu3 levels in RA with high disease-activity DAS28 scores correlated with disease activity measures and may be useful in monitoring disease activities. © 2016 Liou, Huang. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Chen L.-C.,Chang Gung University | Wang L.-J.,Chang Gung University | Tsang N.-M.,Chang Gung Memorial Hospital at Lin Kou | Ojcius D.M.,Chang Gung University | And 8 more authors.
EMBO Molecular Medicine | Year: 2012

Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL-1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG-I inflammasomes are overexpressed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG-I are required for IL-1β induction by EBV genomic DNA and EBV-encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 and NLRP3, respectively. In mice, tumour-derived IL-1β inhibits tumour growth and enhances survival through host responses. Mechanistically, IL-1β-mediated anti-tumour effects depend on infiltrated immunostimulatory neutrophils. We show further that presence of tumour-associated neutrophils is significantly associated with better survival in NPC patients. Thus, tumour inflammasomes play a key role in tumour control by recruiting neutrophils, and their expression levels are favourable prognostic markers and promising therapeutic targets in patients. Inflammasomes detect infection and trigger inflammation. The authors find that in Epstein-Barr virusassociated nasopharyngeal carcinoma, inflammasomes recruit neutrophils, which produce IL-1beta and thereby inhibit tumor growth. © 2012 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.


Li H.-P.,Chang Gung University | Huang H.-Y.,Chang Gung University | Lai Y.-R.,Chang Gung University | Huang J.-X.,Chang Gung University | And 4 more authors.
Oncotarget | Year: 2014

MicroRNAs (miRNAs) play a pivotal role in carcinogenesis by suppressing oncogenes or tumor suppressor genes. Various studies have identified numerous miRNAs and their diverse targets; however, the consequences of dysregulated miRNAs in nasopharyngeal carcinoma (NPC) remain unclear. For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively. Promoter assays demonstrated that upstream stimulatory factor 1 (USF1) is a crucial transcription factor that activates miR-148a promoter activity. EMSA assays confirmed that purified USF1 binds better toward the unmethylated than the methylated CG-containing USF1 consensus probe. The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1. Biochemical and functional assays provided supporting evidence that these 3 genes are the downstream targets of miR-148a in NPC cells. Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression. Overall, our findings revealed that miR-148a can act as tumor suppressor miRNA and serve as a biomarker as well as a therapeutic target for NPC.


Chen L.-C.,Chang Gung University | Chung I.-C.,National Yang Ming University | Hsueh C.,Chang Gung University | Hsueh C.,Chang Gung Memorial Hospital at Lin Kou | And 6 more authors.
Cell Death and Differentiation | Year: 2010

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) mediates antiapoptotic activity in part by inducing downstream antiapoptotic genes. To systematically identify hnRNP K targets in nasopharyngeal carcinoma (NPC), affymetrix chips were used to identify genes that were both overexpressed in primary NPC and downregulated by hnRNP K knockdown in NPC-TW02 cells. The resulting gene set included the antiapoptotic gene, FLIP, which was selected for further study. In cells treated with hnRNP K siRNA, TRAIL-induced apoptosis was enhanced and the FLIP protein level was reduced. Promoter, DNA pull-down and chromatin- immunoprecipitation assays revealed that hnRNP K directly interacts with the poly(C) element on the FLIP promoter, resulting in transcriptional activation. Through iTRAQ-mass spectrometric identification of proteins differentially associated with the poly(C) element or its mutant, nucleolin was determined to be a cofactor of hnRNP K for FLIP activation. Furthermore, FLIP was highly expressed in tumor cells, and this high-level expression was significantly correlated with high-level hnRNP K expression (P=0.002) and poor overall survival (P=0.015) as examined in 67 NPC tissues. A multivariate analysis confirmed that FLIP was an independent prognostic factor for NPC. Taken together, these findings indicate that FLIP expression is transcriptionally regulated by hnRNP K and nucleolin, and may be a potential prognostic and therapeutic marker for NPC.© 2010 Macmillan Publishers Limited. All rights reserved.


Hsu C.-K.,Chang Gung University | Lee I.-T.,Chang Gung University | Lin C.-C.,Chang Gung Memorial Hospital at Lin Kou | Lin C.-C.,Chang Gung University | And 2 more authors.
American Journal of Physiology - Lung Cellular and Molecular Physiology | Year: 2014

Elevated levels of TNF-α have been detected in the airway fluids, which may induce upregulation of inflammatory proteins. Suppressors of cytokine signaling (SOCS)-3 proteins can be induced by various cytokines and negatively regulated inflammatory responses. Although TNF-α has been shown to induce SOCS-3 expression, the mechanisms underlying TNF-α-induced SOCS-3 expression in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we showed that TNF-α induced SOCS-3 expression, which was inhibited by pretreatment with the inhibitor of transcription level (actinomycin D), translation level (cycloheximide), JNK1/2 (SP600125), MEK1/2 (U0126), NADPH oxidase (Nox; apocynin and diphenyleneiodonium chloride), or reactive oxygen species (ROS; N-acetyl-L-cysteine) and transfection with siRNA of JNK1, p47phox, p42, Nox2, or human antigen R (HuR). In addition, TNF-α-stimulated JNK1/2 and p42/p44 MAPK phosphorylation, Nox activation, and ROS generation were inhibited by pretreatment with U0126 or SP600125 and transfection with siRNA of JNK1 or p42. We further showed that TNF-α markedly induced HuR protein expression and translocation from the nucleus to the cytosol, which could stabilize SOCS-3 mRNA. Moreover, TNF-α-enhanced HuR translocation was reduced by transfection with siRNA of p42, JNK1, or p47phox. These results suggested that TNF-α induces SOCS-3 protein expression and mRNA stabilization via a TNFR1/JNK1/2, p42/p44 MAPK/Nox2/ROS-dependent HuR signaling in HTSMCs. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via induction of adhesion molecules and then causes airway and lung injury. Moreover, we also demonstrated that overexpression of SOCS-3 protects against LPS-induced adhesion molecules expression and airway inflammation. © 2014 the American Physiological Society.


Lin Y.-H.,National Taiwan University Hospital | Lin S.-H.,Harvard University | Li P.,Chang Gung University | Li P.,Chang Gung Memorial Hospital at Lin Kou | And 3 more authors.
PLoS ONE | Year: 2013

Background:Phantom vibration syndrome is a type of hallucination reported among mobile phone users in the general population. Another similar perception, phantom ringing syndrome, has not been previously described in the medical literature.Methods:A prospective longitudinal study of 74 medical interns (46 males, 28 females; mean age, 24.8±1.2 years) was conducted using repeated investigations of the prevalence and associated factors of phantom vibration and ringing. The accompanying symptoms of anxiety and depression were evaluated with the Beck Anxiety and Depression Inventories before the internship began, and again at the third, sixth, and twelfth internship months, and two weeks after the internship ended.Results:The baseline prevalence of phantom vibration was 78.1%, which increased to 95.9% and 93.2% in the third and sixth internship months. The prevalence returned to 80.8% at the twelfth month and decreased to 50.0% 2 weeks after the internship ended. The baseline prevalence of phantom ringing was 27.4%, which increased to 84.9%, 87.7%, and 86.3% in the third, sixth, and twelfth internship months, respectively. This returned to 54.2% two weeks after the internship ended. The anxiety and depression scores also increased during the internship, and returned to baseline two weeks after the internship. There was no significant correlation between phantom vibration/ringing and symptoms of anxiety or depression. The incidence of both phantom vibration and ringing syndromes significantly increased during the internship, and subsequent recovery.Conclusion:This study suggests that phantom vibration and ringing might be entities that are independent of anxiety or depression during evaluation of stress-associated experiences during medical internships. © 2013 Lin et al.


Chen C.-C.,Chang Gung University | Liu H.-P.,Chang Gung University | Chao M.,Chang Gung University | Liang Y.,Chang Gung University | And 4 more authors.
Oncogene | Year: 2014

Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is a metastasis-prone epithelial cancer. We previously showed that tumor necrosis factor -induced protein 2 (TNFAIP2) is highly expressed in NPC tumor tissues and is correlated with metastasis and poor survival in NPC patients. However, the underlying mechanism remains unclear. In this study, we demonstrate that the EBV oncoprotein, latent membrane protein 1 (LMP1), can transcriptionally induce TNFAIP2 expression via NF-κB. Quantitative RT-PCR and western blotting revealed that LMP1 induces TNFAIP2 expression through its C-terminal-activating region (CTAR2) domain, which is required for transduction of NF-κB (nuclear factor kappa-light-chain- enhancer of activated B cells) signaling. Inhibition of NF-κB activation or depletion of p65 (a component of NF-κB) by RNA interference abolished the LMP1-induced expression of TNFAIP2, whereas ectopic expression of p65 was sufficient to induce TNFAIP2 expression. Luciferase reporter assays showed that LMP1 transcriptionally induces TNFAIP2 expression through a newly identified NF-κB-binding site within the TNFAIP2 promoter (-3 869 to -3 860 bp). Immunohistochemical analysis of NPC biopsy specimens further revealed a significant correlation between the protein levels of TNFAIP2 and activated p65 (R=0.689, P<0.001), indicating that our findings are clinically relevant. Immunofluorescence microscopy and co-immunoprecipitation assays showed that TNFAIP2 associates with actin and is involved in the formation of actin-based membrane protrusions. Furthermore, transwell migration assays demonstrated that TNFAIP2 contributes to LMP1-induced cell motility. Collectively, these findings provide novel insights into the regulation of TNFAIP2 and its role in promoting NPC tumor progression. © 2014 Macmillan Publishers Limited.


Hsu C.-Y.,Chang Gung University | Yi Y.-H.,Chang Gung University | Chang K.-P.,Chang Gung Memorial Hospital at Lin Kou | Chang Y.-S.,Chang Gung University | And 2 more authors.
PLoS Pathogens | Year: 2014

MicroRNAs (miRNAs) are a family of small RNA molecules that negatively regulate the expression of protein-coding genes and play critical roles in orchestrating diverse cellular processes. This regulatory mechanism is also exploited by viruses to direct their life cycle and evade the host immune system. Epstein-Barr virus (EBV) is an oncogenic virus that is closely associated with multiple human diseases, including nasopharyngeal carcinoma (NPC), which is a highly metastatic type of tumor and is frequently reported in South Asia. Several viral proteins have been found to promote the migration and invasiveness of NPC cells. However, not all tumor tissues express these viral oncoproteins, suggesting that other mechanisms may contribute to the aggressive behavior of NPC tumor cells. A previous sequencing study by our group revealed that the EBV miRNA miR-BART9 was expressed at high levels in all EBV-positive NPC tissues. In the present study, we used gain- and loss-of-function approaches to investigate the effect of miR-BART9 in EBV-negative and EBV-positive NPC cells. We discovered that miR-BART9 promotes the migration and invasiveness of cultured NPC cells. The promigratory activity observed in vitro was manifested as an enhanced metastatic ability in vivo. Computational analysis revealed that miR-BART9 may target E-cadherin, a membrane protein that is pivotal in preserving cell-cell junctions and the epithelial phenotype. Through biochemical assays and functional rescue analysis, we confirmed that miR-BART9 specifically inhibits E-cadherin to induce a mesenchymal-like phenotype and promote the migration of NPC cells. These results indicated that miR-BART9 is a prometastatic viral miRNA and suggested that high levels of miR-BART9 in EBV-positive NPC cells may contribute to the aggressiveness of tumor cells. © 2014 Hsu et al.


Chen C.-C.,Chang Gung University | Chen L.-C.,Chang Gung University | Liang Y.,Chang Gung University | Tsang N.-M.,Chang Gung Memorial Hospital at Lin Kou | Chang Y.-S.,Chang Gung University
Cellular Signalling | Year: 2010

High thymidine phosphorylase (TP) expression is significantly correlated with poor prognosis in patients with nasopharyngeal carcinoma (NPC). NPC is an Epstein-Barr Virus (EBV)-associated cancer in which the EBV-encoded oncogene product, latent membrane protein 1 (LMP1), is expressed in approximately 60% of tumor tissues. However, no previous study has examined whether LMP1 is involved in up-regulating TP expression in NPC tissues. We herein show that LMP1 expression is correlated with TP expression in tumor cells, as examined by quantitative RT-PCR and immunohistochemical staining. We further show that the CTAR1 and CTAR2 domains of LMP1 mediate TP induction, as demonstrated by quantitative RT-PCR and Western blot analyses using LMP1 deletion and site-specific mutants. Mechanistically, LMP1-mediated TP induction is abolished by inhibitors of NF-κB and p38 MAPK, dominant-negative IκB and p38, and siRNA-mediated knockdown of p38 MAPK. Clinically, there were significant correlations among the expression levels of TP, activated p65, and phospho-p38 MAPK in NPC biopsy samples. Functionally, LMP1-mediated induction of TP expression enhanced the sensitivity of NPC cells to the chemotherapeutic prodrug, 5'-DFUR. Our results provide new insights into the roles of LMP1-mediated NF-κB and p38 MAPK signaling pathways in TP induction, potentially suggesting new therapeutic strategies for the treatment of NPC. © 2010 Elsevier Inc.


Chen L.-C.,Chang Gung University | Chen C.-C.,Chang Gung University | Liang Y.,Chang Gung University | Tsang N.-M.,Chang Gung Memorial Hospital at Lin Kou | And 2 more authors.
Modern Pathology | Year: 2011

Tumor necrosis factor alpha (TNFα) is an inflammatory cytokine that is present in the microenvironment of many tumors and is known to promote tumor progression. To examine how TNFα modulates the progression and metastasis of nasopharyngeal carcinoma, we used Affymetrix chips to identify TNFα-inducible genes that are dysregulated in this tumor. Elevated expression of TNFAIP2, which encodes TNFα-inducible protein 2 and not previously known to be associated with cancer, was found and confirmed by quantitative RT-PCR of TNFAIP2 expression in nasopharyngeal carcinoma and adjacent normal tissues. Immunohistochemical analysis showed that the TNFAIP2 protein was highly expressed in tumor cells. Analysis of 95 nasopharyngeal carcinoma biopsy specimens revealed that high TNFAIP2 expression was significantly correlated with high-level intratumoral microvessel density (P0.005) and low distant metastasis-free survival (P0.001). A multivariate analysis further confirmed that TNFAIP2 was an independent prognostic factor for nasopharyngeal carcinoma (P0.002). In vitro, TNFα treatment of nasopharyngeal carcinoma HK1 cells was found to induce TNFAIP2 expression, and siRNA-based knockdown of TNFAIP2 dramatically reduced the migration and invasion of nasopharyngeal carcinoma HK1 cells. These results collectively suggest for the first time that TNFAIP2 is a cell migration-promoting protein and its expression predicts distant metastasis. Our data suggest that TNFAIP2 may serve as an independent prognostic indicator for nasopharyngeal carcinoma. © 2011 USCAP, Inc.

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