| BackgroundGlioma is the most common type of primary malignant brain tumor in adults,accounting for 28% of all primary brain tumors and other tumors of the central nervous system.Gliomas are lethal tumors characterized by diffuse cell infiltration and resistance to chemoradiotherapy.About 56.6% of glioma cases are grade IV astrocytomas,known as glioblastoma(GBM),which are highly aggressive tumors.The prognosis of GBM patients is poor,and the median survival period is only 12-15 months.Currently,the main treatment methods of GBM include surgery,radiotherapy and chemotherapy.Although some achievements have been made with the progress of medical technology,the treatment of GBM is still a big problem due to the high invasiveness of GBM,the uncovered growth and development mechanisms of tumor cells,and the existence of blood-brain barrier.Inflammation is tightly related with the development of tumors.It is associated with various stages of tumorigenesis,such as cell proliferation,tumor transformation,apoptosis escape,loss of cell cycle control,angiogenesis and metastasis.A series of activities of tumor cells depend on tumor microenvironment.An important part of tumor microenvironment is inflammatory factors,and a series of cytokines and chemokines can be produced in the inflammatory response,which plays an important role in promoting tumor invasion and metastasis.Moreover,the accumulation of inflammatory cytokines can promote tumor cells to pass through stromal cells to a large extent,thus leading to the metastasis of tumor cells.Mitochondria are a kind of highly dynamic organelle in eukaryotic cells.Their size,morphology,mobility,localization and turnover are closely related to their adaptability and functional changes,quality control and homeostasis.Mitochondria exhibit a long filamentous network structure,and as they move in all directions throughout the cytoplasm,they can be seen to extend,contract,break and fuse,and this feature is necessary to maintain mitochondria-related biological activities,such as cell growth,division,apoptosis,aging and disease.Mitochondria are directly or indirectly involved in many aspects of tumor cell metabolism.It has been reported that mitochondrial changes are involved in various solid tumors and hematologic malignancies.Although many studies have proposed multiple mechanisms to explain the mitochondrial dynamics of tumor cells,there were no studies assessing the effect of inflammation on glioma mitochondria.Therefore,it is necessary to explore the functional and morphological changes of mitochondria in glioma to understand the occurrence and development of glioma.Therefore,we applied the bioinformatics,transmission electron microscopy,cell culture,immunofluorescence and flow cytometry,immunohistochemistry and ELISA,to study the morphology and biology of mitochondria in glioma,as well as the effects of inflammation on the mitochondria of glioma cell.Materials and MethodPart I.Mitochondria and inflammation in glioma tissue1.Transmission electron microscope was used to examine the ultrastructure of mitochondria in glioma tissues.2.We download the glioma data from the Chinese Glioma Genome Atlas(CGGA)dataset and analyzed the gene expression of enzymes related to glycolysis,electron transport chain and tricarboxylic acid cycle and patient survival analysis;we also analyzed the gene expression of inflammation related factors in glioma and their correlation with the expression of enzymes related metabolic.3.HE staining was performed on glioma tissues and adjacent tissues to observe the infiltration of inflammatory cells in glioma tissues.Besides,Iba1 immunohistochemistry was performed to examine the reactivation of microglia cells.4.According to the protein expression information in glioma provided in The Human Protein Atlas,the expression changes of each subunit of the four electron transport chain complexes in mitochondria in glioma were statistically analyzed.Part II.The effect of inflammation on mitochondrial dynamics in glioma cells1.LPS and IFN-? were used to induce the inflammatory response in glioma cells.The stimulating time course were 0 h,4 h,8 h and 24 h.Firstly,the inflammatory cytokines IL-6,IL-1?,TNF-? and IL-4 were examined with ELISA.Then,the immunofluorescence and transmission electron microscopy were used to observe the morphological changes of mitochondria in glioma cells.Western blot was applied to test the expression level of Drp1,Mfn2 and Hsp60.2.MitoSOX-Red was used to detect the changes of mtROS in glioma cells.3.Mitochondrial membrane potential detection kit(JC-1)was used to detect the changes of mitochondrial membrane potential in glioma cells.4.Western blot was used to detect the protein expression of enzymes related to glycolysis,electron transport chain and tricarboxylic acid cycle in glioma cells.5.LC 3B was used to label autophagosome.Western blot and immunocytochemistry were used to detect the changes of autophagy level in glioma cells.6.The viability of glioma cells was detected with CCK8,and Ki-67 labeled proliferating cells were used to detect proliferation of glioma cells,and the apoptosis of glioma cells was detected with Annexin V FITC/ PI.Results1.Mitochondrial swelling,matrix vacuolation,and cristae destruction were observed in glioma tissues under transmission electron microscope.2.According to the statistical analysis of glioma data in CGGA dataset,the expression of glycolytic enzymes HK1,LDHA and GAPDH was increased in GBM,and their overexpressions were negatively related with the survival of patients;The gene expressions of ATP5 A and CS,which are related to electron transport chain and tricarboxylic acid cycle,were decreased in GBM,and the downregulation of ATP5 A and CS could reduce the survival rate of patients.The expression of various inflammatory factors in GBM was also changed.The inflammatory factors IL-6,IL-8,IL-4 and IL-10 were positively correlated with the expression of glycolytic enzymes HK1,LDHA and GAPDH,and negatively correlated with the expression of electron transport chain and tricarboxylic acid cycle-related enzymes ATP5 A and CS.3.Necrotic core was observed in GBM tissues by HE staining.Besides,the infiltration of inflammatory cells and the reactivation of microglia cells could also be observed in GBM tissues by HE and IHC staining.4.The protein expressions of many subunits of the four electron transport chain complexes in the mitochondria were obviously changed in glioma tissues.Immunohistochemical staining results revealed significant change of COX6B1,Subunit 6 and SDHB in glioma tissues.5.After the LPS+ IFN-? stimulation,we acquired the following results in glioma cells: Firstly,the expressions of proinflammatory factors IL-6,IL-1? and TNF-? were increased,while the expression of anti-inflammatory factor IL-4 was not significantly changed or decreased.Second,the fragmentation of U118-MG mitochondria was increased.And the fragmented mitochondria appeared as the spherical,annular or Cshaped morphology.Third,the ultrastructure of mitochondria was damaged: swollen,vacuolated matrix,disrupted cristae,and C-shaped morphology.Finally,western blot test showed that expression of Drp1 was increased and expression of Mfn2 was decreased.6.Stimulation of LPS+ IFN-? lead to the increase of mtROS in U87-MG at 4h and 8 h,and there was no significant change of mtROS in U118-MG.The membrane potential was decreased in two kinds of glioma cells.7.HK1 and LDHA,which were enzymes related to glycolysis,were significantly increased at 8 h after LPS+ IFN-? stimulation in with U87-MG glioma cells.CS,which was related to the tricarboxylic acid cycle,was significantly increased after stimulation.Electron transport chain complex subunit COX6B1 and SDHB were significantly decreased at 8 h and 24 h after stimulation,while ATP5 A was not significantly changed.8.Stimulation with LPS+ IFN-? did not changed the level of autophagy,cell activity and proliferation,while the apoptosis was significantly decreased.Conclusions1.In glioma tissues,the mitochondria are disrupted,reprogramming of metabolism occurs,and inflammatory response exists,and there is a correlation between metabolism and inflammation.2.Stimulation of proinflammatory can destroy the structure of mitochondrial,and increase mtROS,and decrease mitochondrial membrane potential in glioma cells;glioma cells are resistant to inflammatory mitochondrial abnormalities. |
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