Expression Of HMGB1 In Gliomas And Its Effect On Glioma Cells

Background and Aims:High mobility group box 1(HMGB1)is a noval non-histone chromatin protein that expressed in eukaryotic cells.HMGB1 participates gene transcription,DNA repair,V(D)J recombination,extracellular signal transduction,construction and stability of nucleosome structure,cell proliferation,differentiation,migration and apoptosis,tumor occurrence,growth,metastasis and invasion.It contains 215 amino acid residues and is highly conserved during evolution.HMGB1 is consisted of two HMG box domains and a C terminal of 30 amino acids.HMGB1 could regulate translation,DNA repair and recombination through remodeling the chromatin by combining DNA.HMGB1 could be released out of the cell when the cells were stimulated,cell death,apoptosis,tissue hypoxia and ischemia reperfusion.Realeased HMGB1 plays a role in inflammation,migration,differentiation and regeneration.In addition,HMGB1 also has a wide range of regulatory roles in cytokine release,cell proliferation,angiogenesis,and many other aspects.In recent years,many studies have shown that the mechanism of HMGB1 involving in the development and progression of tumors might be that high expression of HMGB1 leads to the abnormal expression of some genes,resulting in the phenotype of tumor cells.Besides,the high expression of HMGB1 could protect those cells from apoptosis,leading to the occurrence of tumor.HMGB1 was highly expressed in the tumor tissues of colon cancer,prostate cancer,lung cancer and esophageal cancer.And investigators also found the expression of receptor for advanced glycation end products(RAGE)in those tumor tissues.As the receptor of HMGB1,RAGE could influence the the expression of HMGB1 via JAK/STAT pathway.Glioblastoma(GBM)is the most common malignant tumor of the brain with a high mortality rate because of high invasion and metastasis.Glioma pathological type could be divided into four grades according to the standards set by the WHO: grade I,pilocytic astrocytoma;grade II,diffuse astrocytoma,grade III,anaplastic astrocytoma;grade IV,glioblastoma multiforme.Grade I and grade II are benign tumors belonging to the low level.While grade III and grade IV are malignant tumors belonging to the high level.Although the treatment of glioma has been made great progress in surgery,chemotherapy and radiotherapy recent years,the effectiveness of treatment is greatly limited since the invasion of tumor cells,blood brain barrier,uncertainty of the source and growth mechanism of tumor cells.There is a study exploring the relationship between HMGB1 and brain glioma by examining the m RNA expression levels of HMGB1.And the results showed that the m RNA expression levels of HMGB1 in brain glioma tissues were significantly higher than that in controls tissues.While the expression levels of HMGB1 between low-grade gliomas and high-grade gliomas had no significant differences.This results show a strong relationship between HMGB1 and brain glioma.Kuniyasu et al.found that 65% RAGE positive and 85% HMGB1 positive in 96 patients with gastric carcinoma.Other studies indicated that HMGB1 could be released by tumor cells with high expression of HMGB1 or necrotic tumor cells,promoting the proliferation of adjacent tumor cells and inducing the regeneration of small blood vessels.It was also found that the inhibitor of HMGB1 or RAGE could inhibit the proliferation of some tumor cells.To date,the specific effect and mechanisms of HMGB1 on glioma was not clear.For example,the expression levels of HMGB1 in different grade gliomas,the location of HMGB1 in cells,the location of HMGB1 in which cell types,the effects and mechanisms of HMGB1 on glioma cell proliferation,survival,apoptosis and autophagy,all those questions need to be explored.The aim of the present study was to illustrate the expression level,cellular localization and expressed cell types of HMGB1 in various grades of gliomas and to explore the influences of overexpressed HMGB1 on mitochondrial morphology,cell apoptosis and autophagy in glioma,providing a theoretical basis for the clinical therapy of glioma.Materials:Paraffin sections of various grades of gliomas were provided by the department of pathology in the 153 rd central hospital of Chinese People’s Liberation Army.All the specimens were obtained from the surgical resection of glioma patients in the department of neurology of the hospital,including 12 cases of low-grade glioma specimens and 15 cases of high-grade glioma specimens.The cell lineused in cell experiments were U87-MG glioma cells bought from the cell bank of Chinese Academy of Sciences.Methods: 1.Paraffin section immunohistochemical staining for HMGB1: to dectect the expression level,cell location,expression cell types of HMGB1.2.Plasmid construction and extraction.HMGB1 over-expressed plasmid was synthetized by Sangon Biotech(Shanghai).HMGB1 coding sequence was inserted in the p EGFP-C1 plasmid,which makes fused expression of HMGB1 and EGFP.p EGFP-C1 vacant vector served as the control plasmid.Plasmids were extracted with BIOMIGA Extraction Kit.3.Cell transfection: Simple-Fect Transfection Reagent was used to transfect the plasmids into U87-MG cells.Then we detected the HMGB1 overexpression with WB and immunocytochemistry methods.And we use immunocytochemistry to observe the influence of HMGB1 overexpression on the morphology of mitochondria,cell apoptosis and autophagy.4.U87-MG cells were stimulated with LPS&IFN-γ.Then the expression level of HMGB1 was observed by WB.5.The statistical analysis was performed with the SPSS 17.0 software(SPSS Inc.,Chicago,IL,USA)and origin 70.The data were demonstrated with Mean ± SE.The difference analysis among different groups was performed using One-way ANOVA.Results: 1.The percentage of high-expressed HMGB1 in high grade glioma specimens was significantly higher than that in low grade glioma specimens(χ2 = 7.56,P = 0.01).There was no significant difference between high grade glioma specimens and low grade glioma specimens for the percentage of moderate-expressed HMGB1 specimens(χ2 = 1.17,P = 0.18)and the percentage of low-expressed HMGB1 specimens(χ2 = 1.93,P = 0.13).2.The positive signals of HMGB1 were mainly located in the nucleus.Only few proportion of cells have the cytoplasm location of HMGB1.Statistical analysis showed that in all gliomas,the cell percentage of HMGB1 located in the nucleus were significantly higher than the cell percentage of HMGB1 in cytoplasm(low grade P = 0.00797;high grade P = 0.0013).The cell percentages of HMGB1 expressed in the nucleus of the two grades gliomas has no significant difference,as well as the cell percentages of HMGB1 expressed in the cytoplasm(low grade P = 0.79254;high grade P = 0.44251).In addition,the expression of HMGB1 appears irregular or lobulated morphology in the high grade glioma tissues.However,the expression of HMGB1 demonstrates the round or oval phenotype in the low grade glioma tissues.3.In glioma tissues,HMGB1 was expressed in glioma associated astrocytes(GFAP labeling),glioma stem cells(Nestin labeling),microglia cells(Iba1 labeling)and vascular endothelial cell-like cells and neuron-like cells.4.The microglia cells in high-grade gliomas showed a markedly activated state(neurite retraction,soma enlargement and roundness).In addition,the expression of inflammatory factor IL-6 was significantly increased(P = 0.01780).The expression level of HMGB1 was increased after the stimulation of LPS and IFN-γ in U87-MG cells.5.HMGB1 is mainly expressed in the nucleus in U87-MG cells,with the more intensify staining in nucleoli.6.Overexpression of HMGB1 induced the fragmentation of mitochondria in U87-MG cells.And we did not find the influence of HMGB1 overexpression on the expression of LC3 B and Caspase3 in U87-MG cells.Conclusion: 1.The HMGB1 expression is increased in high grade gliomas;It mainly loacates in the nulear;It is expressed in glioma-associated astrocytes,glioma stem cells,microglia cells,vascular endothelial cell-like and neuron-like cells.2.Inflammation causes the increase of HMGB1 expression in glioma cells.The positive signal mainly locates in nulear of glioma cells.Overexpression of HMGB1 leads to the mitochondria fragmentation.