Subcellular Localization Of HMGB1 In Gliomas And The Function Of Its Domains In Localization

Background and PurposeGlioma is the most common primary tumor of the central nervous system(CNS).It can be divided into low grade glioma(LGG)and high-grade glioma(HGG)in histopathology.Glioblastoma(GBM)is the most common type of HGG,Which has the characteristics of "three highs and one low" : high morbidity,high recurrence,high mortality,and low cure rate.Traditional treatments such as drug therapy,surgical resection,and radiotherapy and chemotherapy have not significant effects on the treatment of high-grade gliomas.The high mobility group box 1 protein(HMGB1)is a very conserved non-histone protein and binding DNA in eukaryotic cells.Under normal cell growth conditions,HMGB1 is mainly located in the nucleus.When the cells are subjected to starvation,hypoxia-ischemia,oxidative stress or radiotherapy and chemotherapy,HMGB1 can be transferred from the nucleus to the cytoplasm and be secreted to extracellular space.At present,the results of research on the subcellular structure localization of HMGB1 in the cytoplasm are not consistent.In hypoxic-stressed neurons,HMGB1 can be localized on mitochondria and peroxisomes;in monocytes/macrophages,HMGB1 localizes on endosomes and lysosomes,and is secreted to the extracellular space through the endosome-lysosome pathway;in HEK293 T and mouse embryonic fibroblasts,HMGB1 localize on autophagosomes and is secreted to the extracellular space though the non-classical pathway mediated by the early autophagy(HMGB1lacks signal peptide and cannot be secreted to the outside of the cell through the classical pathway mediated by the endoplasmic reticulum-Golgi apparatus).In different cells,HMGB1 localizes in different subcellular structures,and the pathway of extracellular secretion is different as well.A number of studies have shown that the translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular secretion lead to tumor autophagy,inflammation,cell proliferation and invasion.Inhibition of HMGB1 cytoplasmic translocation and extracellular secretion has become a treatment strategy.At present,the specific subcellular localization of HMGB1 in glioma has not been elucidated.Exploring the subcellular localization of HMGB1 in gliomas will help to provide new theoretical support for the secretion pathway of HMGB1 to extracellular in gliomas,and provide new targets and strategies for the treatment of gliomas!Method1.The glioma cell U87-MG were stimulated through Hank’s balanced salt solution(HBSS)to induce the transfer of HMGB1 from the nucleus to the cytoplasm and the extracellular secretion.The immunofluorescence,nuclear and cytoplasmic proteins separation,Western Blot(WB)、Enzyme-Linked Immuno Sorbent Assay(ELISA)were used to examine the nuclear and cytoplasmic transfer and extracellular secretion of HMGB1 in glioma cells.2.The potential interacting proteins of HMGB1 were prediced through bioinformatics,and then the subcellular localization of those proteins was analyzed.3.The subcellular localization of HMGB1 in U87-MG cells was detected through the double immunofluorescence staining.furthermore,we also examined the alteration of HMGB1 subcellular localization during the different stimulating time points.4.The immunoelectron microscopy was also used to detect the subcellular localization of HMGB1 in glioma cells.5.We constructed different HMGB1 mutants plasmids,including A Box,B Box,ΔC(delete C-tail),ΔNLS1(delete NLS1)and ΔNLS2(remove NLS2),and transfect them into U87-MG glioma cells,then detect the subcellular localization of each mutant of HMGB1 in U87-MG by double immunofluorescence staining.Result1.Expression of HMGB1 in the cytoplasm was significantly increased and reached to a maximum value after 1 h of HBSS stimulation.Moreover,the extracellular secretion of HMGB1 reached to the maximum after 2 h of stimulation.2.The proteins that potentially interact with HMGB1 were localized in nucleus,cytoplasm,and extracellular space.In cytoplasm,those proteins can be localized in mitochondria,endoplasmic reticulum,lysosomes,Golgi complex,endosomes,autophagosomes and cytoskeleton.3.The results of double immunofluorescence staining and immunoelectron microscopy indicated that HMGB1 localized in the nucleus,cytoplasm and extracellular space of the HBSS stimulated U87-MG cells.In the cytoplasm,HMGB1 localized in mitochondria,endoplasmic reticulum,autophagosomes,early endosomes,late endosomes,lysosomes and cytoskeletons.Compared with HBSS stimulation for 1h,the localization of HMGB1 in autophagosomes,late endosomes,lysosomes,mitochondria and peroxisomes was significantly reduced,the localization on the early endosome and endoplasmic reticulum did not change significantly,while the localization on the cytoskeleton was significantly increase after HBSS stimulation for3 h.4.The results of HMGB1 mutants assay demonstrated thatthat the subcellular localization of each mutant was different in U87-MG cells.A Box is mainly located on autophagosomes and early endosomes;B Box is mainly located on peroxisomes,endosomes and plasma;ΔC is mainly located on autophagosomes and early endosomes;ΔNLS1 and ΔNLS2 are mainly located on peroxisomes and early endosomes.Meanwhile,the mutants located on lysosomes are mainly A Box and B Box;the mutants located on the autophagosome are mainly A Box and ΔC,the mutants located on the mitochondria are mainly A Box and B Box,and the mutants located on the early endosome are mainly A Box and ΔNLS1;the mutants located on the endosome are mainly A Box and ΔC,the mutants located on the endoplasmic reticulum are mainly A Box and B Box,and the mutants located on the peroxisome are mainly A Box and B Box.ConclusionHMGB1 translocates from nucleus to cytoplasm and is secreted to extracellular space in response to starvation stress.The functional domains of HMGB1 mediate its location on the cytoskeleton and endomembrane system..