3146

HMGB1

BACKGROUND: high-mobility group box protein 1 (HMGB-1) is a chromatin-binding protein that bends DNA, thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. METHODS: we established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMCs). The model was characterized using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. RESULTS: activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased alpha-smooth muscle actin expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products and through induction of transforming growth factor-beta(1) secretion from PTECs. CONCLUSIONS: these results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis.CI - 2010 S. Karger AG, Basel.

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial-to-mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT-promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high-mobility group box-1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin-8 (IL-8) at early-stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV-SPORT6-HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU-1 and KATOIII), N87-xenograft BALB/c nude mice and human paired serum-tissue GC samples. HMGB1-induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL-8 were evaluated in N87-xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1-induced IL-8 overexpression contributed the HMGB1-induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL-8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL-8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL-8 as a promising diagnostic and therapeutic target to control GC micrometastasis.CI - (c) 2015 UICC.

epithelial-to-mesenchymal transition (EMT) is a crucial event in the cellular origin of myofibroblasts that secrete extracellular matrix in the progression of pulmonary fibrosis (PF). High-mobility group box 1 (HMGB1) is a novel mediator of EMT. However, whether this process involves the recognized transforming growth factor-beta1 (TGF-beta1)/Smad signaling that also contributes to EMT in PF has not yet been elucidated. Here, we developed a model of PF induced by bleomycin (BLM) in rats and conducted several simulation experiments in A549 (human) and RLE-6TN (rat) alveolar epithelial cell (AEC) lines to unravel the role of TGF-beta1/Smad2/3 signaling in HMGB1-mediated EMT. We found that the levels of serum HMGB1 and lung hydroxyproline were severely elevated after BLM administration. Moreover, the protein expression of HMGB1, TGF-beta1, phosphorylated Smad2/3 (p-Smad2/3), and mesenchymal markers including alpha-smooth muscle actin, vimentin, and type I collagen were significantly increased with the reduced protein expression of an epithelial marker (E-cadherin) in the rat model by Western blot or immunohistochemical analysis. In addition, the uptake of both exogenous TGF-beta1 and HMGB1 by AECs could induce EMT; meanwhile, HMGB1 dramatically enhanced TGF-beta1 expression and triggered Smad2/3 phosphorylation. In contrast, TGF-beta1 deficiency evidently ameliorated HMGB1-mediated EMT with reduced p-Smad2/3 in A549 cells. It provides new insights that HMGB1 release from injured lungs promotes AEC damage through induction of the EMT process, in which TGF-beta1/Smad2/3 signaling is activated and contributes to PF. These results suggest that HMGB1 may constitute a therapeutic target for developing antifibrotic agents for abnormal lung remodeling.CI - Copyright (c) 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Highmobility group box 1 (HMGB1), a member of the highmobility group protein family, was originally characterized as a nonhistone, nuclear DNAbinding protein. While the roles of HMGB1 in inflammation and cell differentiation have been previously reported, its role in tumor cell migration and invasion, particularly in hepatocellular carcinoma (HCC), has remained elusive. The present study reported that the expression of HMGB1 in HCC tissues was significantly higher than that in matched tumoradjacent tissues (P<0.05). HMGB1 was expressed at significantly elevated levels in tumors of patients with large tumor size, high histological grade and advanced tumornodemetastasis stage (P<0.05). The positive expression of HMGB1 correlated with a poor threeyear overall and diseasefree survival of HCC patients (P<0.05). In addition, HMGB1 was an independent factor for predicting the threeyear overall and diseasefree survival of HCC patients (P<0.05). An in vitro experiment revealed that knockdown of HMGB1 inhibited cell migration and invasion in the HCC cell lines Huh7 and MHCC97H (P<0.05). Furthermore, western blot analysis showed that HMGB1 knockdown markedly inhibited epithelial mesenchymal transition in Huh7 and MHCC97H cells. These results suggested that HMGB1 may be utilized as an independent prognostic marker in HCC and may promote tumor progression by promoting cell migration and invasion.

epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3beta inactivation, cytoplasmic accumulation, and nuclear translocation of beta-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and beta-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3beta/beta-catenin signaling pathway.

Abnormal expression of high-mobility group box-1 (HMGB1) protein occurs in many tumors and is closely associated with tumor invasion and metastasis. However, a role for HMGB1 in epithelial-mesenchymal transition (EMT) in hypopharyngeal carcinoma has not been previously reported. We cultured cells of the hypopharyngeal carcinoma cell line FaDu in vitro and then treated them with 5 ng/ml TGF-beta1 for 48 h to induce EMT. Vimentin, Snail, and HMGB1 expression patterns were then detected using immunofluorescence staining; HMGB1 mRNA and protein expression were verified by RT-PCR and western blot analyses. HMGB1 was then silenced in FaDu cells using RNAi, followed by detection of Vimentin, Snail, and HMGB1 expressions by immunofluorescence staining. The mRNA expression levels of Vimentin, Snail, HMGB1, and E-cadherin were verified by RT-PCR, while protein expression of HMGB1 and receptor for advanced glycation end products (RAGE) were detected by western blot analysis. The biological behavior of FaDu cells was observed before and after HMGB1 silencing using wound healing and cell invasion assays. Following culture with 5 ng/ml TGF-beta1 for 48 h, the morphology of FaDu cells changed from a regular cobblestone-like appearance into a spindle-like shape. expression levels of Vimentin, Snail, and HMGB1 were upregulated at both mRNA and protein levels as determined by RT-PCR, immunofluorescence, and western blotting. After HMGB1 silencing, mRNA expression levels of the epithelial cell marker E-cadherin were upregulated. Meanwhile, expression levels of the mesenchymal markers Vimentin and Snail were decreased. Western blotting revealed that HMGB1 and RAGE were downregulated. RNAi-mediated inhibition of HMGB1 expression decreased the capacities of FaDu cells for invasion and metastasis as determined by wound healing and cell invasion assays. HMGB1 is essential for maintaining the interstitial cell phenotype in TGF-beta1-induced EMT of FaDu cells, and silencing HMGB1 greatly inhibits the invasive and metastatic ability of these cells.

Previous literatures have reported the role of human micro RNA-1284 (hsa-miR-1284, in short miR-1284) in diverse cancers. However, its biological function in osteosarcoma pathogenesis remains unknown. In the present study, we investigated the potential role of miR-1284 in osteosarcoma. expression of miR-1284 and high mobility group box 1 (HMGB1) were examined in 80 tissues obtained from 40 patients. MiR-1284 level was measured in five osteosarcoma cell lines. Relative luciferase activity and HMGB1 expression were examined in MG-63 and U2OS cells transfected with wild-type or mutant 3'-UTR of HMGB1 in the presence of miR-1284 mimics or miR-NC. Cell viability, colony formation, and cell migration were measured in MG-63, U2OS and hFOB 1.19 cells, which were transfected with miR-1284 mimics or miR-NC. In the rescue experiments, recombinant HMGB1 plasmid was transfected into MG-63 and U2OS cells, and cell viability and migration were determined again. Our results indicated that relative level of miR-1284 was lower in tumor tissues compared with its adjacent tissues and it was found suppressed at lower levels in MG-63 and U2OS cell lines. expression of HMGB1 is significantly elevated in tumor tissues and negatively correlated with miR-1284 expression. MiR-1284 exerted its function by directly binding to 3'-UTR of HMGB1 and regulates expression of HMGB1. The overexpression of miR-1284 inhibited the cell proliferation and migration, and altered the protein expression of epithelial-mesenchymal transition (EMT)-associated genes (E-cadherin, N-cadherin, Vimentin, and Snail), which was reversed by HMGB1 overexpression. In conclusion, miR-1284 can function as a new regulator to inhibit osteosarcoma cell proliferation and migration by targeting HMGB1.CI - (c) 2018 The Author(s).

OBJECTIVE: This study aims to investigate the role of NAC1/HMGB1 signaling pathway in the epithelial-mesenchymal transition (EMT), invasion, and metastasis of lung cancer cell line. METHODS: Human lung cancer cell line A549 was used in this study. They were randomly divided into normal control group, sh-NAC1 empty vector group (sh-NAC1 NC), expression empty vector group (NAC1 NC), NAC1-shRNA and NAC1 over-expression group (NAC1). NAC1 and HMGB1 expression levels were detected by qRT-PCR method. Cell proliferation was detected by CCK8 kit. Cell cycles were detected by flow cytometry method. Cell invasion was detected by Transwell method. The expression levels of E-cadherin, N-cadherin, NAC1, and HMGB1 were detected by qRT-PCR and Western blotting methods. RESULTS: Compared with the control group, the expression level of NAC1 and cell proliferation in NAC1-shRNA group decreased, cells in G1 phase increased and cells in S phase decreased. In NAC1-shRNA group, E-cadherin expression levels increased and the expression levels of N-cadherin, HMGB1 and Vimentin decreased. In NAC1 group, the expression level of NAC1 and cell proliferation increased, cells in S phase increased and cells in G1 phase decreased, E-cadherin expression levels decreased and the expression levels of N-cadherin, HMGB1 and Vimentin increased. All these differences are statistically significant. CONCLUSIONS: The expression of NAC1 and HMGB1 in lung cancer cells may affect the occurrence of EMT, the NAC1/ HMGB1 signaling pathway is associated with the EMT, invasion, and metastasis of lung cancer cells.CI - (c) 2018 by the Association of Clinical Scientists, Inc.