| BackgroundGlioma,with distinct characteristics,is the most common and malignant primary tumor of the central nervous system in adults.Glioma accounts for about seventy percent of brain tumors.Moreover,over fifty percent of gliomas are glioblastomas multiforme(GBM),which are the most malignant gliomas.Despite advances in surgical techniques,radiotherapy,chemotherapy,immunotherapy and therapies targeting mechanisms intrinsic to gliomas,the multiple anti-glioma treatments have not reached the satisfactory effects in clinical.Glioma is always the malignant brain tumor with low cure rate,high recurrence rate and poor prognosis.The recalcitrance of malignant gliomas to standard therapy and other therapy strategies is due to the ability of gliomas to invade diffusely into brain parenchyma and the unique tumor microenvironment(TME)which can lead to immunosuppression and therefore facilitate glioma progression.Hence,by doing research on glioma immunosuppressive microenvironment and glioma malignant phenotypes,we can reveal the mechanisms of glioma progression,therefore providing novel anti-glioma therapy strategy and improving anti-glioma treatment effects.The tumor microenvironment is a complex system which formed during the progression of tumors.It is composed of diverse factors and cells and plays important roles in facilitating tumor progression,promoting chemoresistance and resulting in immunosuppression.Because of the rapid proliferation of tumors and abnormal structure and function of blood vessels in the tumor microenvironment,hypoxia becomes a well-characterized feature of the solid tumor microenvironment.As a kind of solid tumor,glioma has hypoxic area in its microenvironment.Hypoxia can regulate glioma malignant behaviors,leading to the switches in metabolism and more aggressive phenotypes.Furthermore,hypoxic can facilitate the generation of glioma immunosuppressive microenvironment.Hence,it will help find new targets and improve anti-glioma therapy to demonstrate the mechanisms under which hypoxia facilitates glioma progression and promotes the formation of the glioma immunosuppressive microenvironment.As an important component of the tumor microenvironment,exosomes play crucial roles in tumor progression.Exosomes are small membrane vesicles,which are 30-100 nm in size.They can be secreted by multiple kinds of cells and contain mRNAs and proteins,therefore participating in intercellular communication by delivering their contents.In the tumor microenvironment,exosomes can target tumor cells to promote tumor progression.In addition,exosomes can also target the immune cells to result in immunosuppression.Moreover,hypoxia can enhance the release and alter the content of exosomes,therefore influencing exosomes to modulate their target cells.Hence,it will help understand the mechanisms of glioma progression and immunosuppression and providing novel foundation for anti-glioma therapy strategy to investigate how the hypoxia influences the glioma malignant behaviors and immunosuppressive microenvironment by exosomes.The immune cells which reside in and around the tumor microenvironment include tumor associated macrophages(TAM),myeloid-derived suppressor cells(MDSC),regulatory T cells(Treg)and regulatory dendritic cells(regDC),all of which are important factors of immunosuppressive tumor microenvironment.Moreover,tumor associated macrophages are the most abundant infiltrating immune cells in the glioma microenvironment.Two polarized macrophage phenotypes have been identified,including classically activated macrophages(M1 type)and alternatively activated macrophages(M2 type).Tumor associated macrophages are more likely to become M2-type macrophages and involved in the immunosuppressive tumor microenvironment.Hypoxia can induce macrophage M2 polarization,but it is not clear whether exosomes participate in the hypoxia-induced macrophage polarization.Hence,it will help find novel targets and evidence for preventing glioma progression and promoting anti-glioma treatment to reveal the mechanisms under which hypoxia induce macrophage M2 polarization by glioma exosomes.Besides immunosuppressive tumor microenvironment,glioma malignant behaviors also lead to the failure of anti-glioma treatment.Migration and invasion are important hallmarks of gliomas.It is the uncontrollable invasiveness that limits the anti-glioma treatment effect and results in the tumor recurrence.The process of migration and invasion include extracellular matrix(ECM)degradation,actin cytoskeleton-based tumor cell movement and epithelial-mesenchymal transition(EMT),which involves matrix metalloproteinases and Rho proteins and is regulated by PI3K and MAPK signaling pathways.Hypoxia can promote the expression of MMP,participate in regulating cytoskeleton and induce EMT,therefore enhancing glioma migration and invasion,but it is still unknown whether glioma exosomes are involved in these procedures.Hence,it will help find new targets for preventing glioma progression and improving anti-glioma treatment to demonstrate the mechanisms under which hypoxia promotes glioma migration and invasion by glioma exosomes.Among the genes and proteins which can exactly influence the glioma migration and invasion,the Rho family of GTPases,including RhoA,Racl and Cdc42,are key mediators of glioma migration and invasion.Moreover,guanine nucleotide exchange factors(GEF)are the key regulators of Rho GTPases.Pleckstrin homology and RhoGEF domain containing G5(PLEKHG5),which belongs to the GEFs,can regulate Rho GTPases and participate in cell migration,cell autophagy and angiogenesis.However,the expression pattern and function of PLEKHG5 in gliomas are still not clear.Hence,it will help find novel target for anti-glioma treatment and improve patients' prognosis to determine the expression pattern and function of PLEKHG5 in gliomas.In our study,focusing on glioma immunosuppressive microenvironment and glioma malignant phenotype,we demonstrated the mechanisms under which the hypoxic glioma exosomes induced macrophage M2 polarization,the mechanisms under which the hypoxic glioma exosomes promoted normoxic glioma migration and invasion,and determined the expression pattern and function of PLEKHG5 in gliomas.In conclusion,our study revealed novel findings in glioma immunosuppressive microenvironment and glioma malignant behaviors.We provide new foundation to understand the mechanisms of glioma progression and find novel targets for anti-glioma treatment,which can help improve treatment effect and have important academic interest and bright prospect.Part ?:The effect and mechanism of hypoxic glioma exosomes on macrophage M2 polarization1.Objective(1)To determine the effect of hypoxic glioma exosomes on macrophage M2 polarization.(2)By performing microRNA sequencing on glioma exosomes,to determine the microRNAs which are enriched in hypoxic glioma exosomes and may be associated with macrophage M2 polarization(microRNA-1246).(3)To determine the effect of microRNA-1246 on macrophage M2 polarization and demonstrate the mechanisms under which microRNA-1246 influence macrophage M2 polarization.(4)To reveal the expression pattern of microRNA-1246 in glioma patients'cerebrospinal fluid(CSF)and determine the clinical relevance of microRNA-1246 in glioma patients.2.Method(1)Isolation and identification of exosomesBy ultracentrifugation and precipitation methods,we isolated exosomes from supernatants of glioma cell lines.The exosomes were identified by transmission electron microscope(TEM),qNano analysis and Western blot.(2)Evaluation of macrophage M2 polarizationThe methods to evaluate macrophage M2 polarization are as below:?M2-type macrophage markers(CD 163,IL10,IL1RA and so on)expression were examined by PCR.?Flow cytometry was used to determine the expression of CD 163 on macrophage surface.?The levels of cytokines(IL-10 and TNF-?)were detected by ELISA.(3)Determination of the glioma proliferationAfter the co-culture of glioma cells and treated macrophages,EdU assay was used to determine the proliferation of glioma cells.(4)Determination of the glioma migration and invasionAfter the co-culture of glioma cells and treated macrophages,Trans well assay was used to determine the migration and invasion of glioma cells(5)MicroRNA sequencing of glioma exosomesAfter exosome isolation,microRNA sequencing was performed to determine the expression pattern of microRNAs in normoxic and hypoxic glioma exosomes.(6)MicroRNA sequencing of glioma patients' cerebrospinal fluidAfter the glioma patients' cerebrospinal fluid collected,microRNA sequencing was performed to determine the expression pattern of microRNAs in glioma patients'cerebrospinal fluid.(7)Detection of microRNA,mRNA and protein levelsSpecial treatments were used to extract the total RNA or proteins from the exosomes or treated cells,and PCR and Western blot were used to detect the expression levels of microRNAs,mRNAs and proteins.(8)Cell transfection and construction of microRNA-1246-expressing U937 cell linesMicroRNA-1246 mimics,microRNA-1246 inhibitors,TERF2IP-overexpression plasmids,TERF2IP siRNAs,luciferase reporter plasmids containing mutant or wild-type TERF2IP 3'UTR binding domain were used in cell transfection with Lipo3000.The purpose of cell transfections was to knockdown or overexpress target molecules to study the phenotype changes.The microRNA-1246 overexpression lentivirus were used to transfect the U937 cell lines to construct microRNA-1246-expressing U937 cell lines.(9)The mRNA sequencing of macrophagesThe mRNA sequencing was performed on control and microRNA-1246-expressing macrophages to determine the expression pattern of mRNAs in different treated macrophages.(10)Models of human GBM in nude mice by orthotopic transplantationLuciferase-labeled U87MG glioma cells mixed with different conditioned macrophages were co-injected into the brains of nude mice.Bioluminescence imaging was used to image the tumors and nude mice were followed up every day.Tumor samples from xenograft models were used for the further studies.3.Results(1)Hypoxic glioma exosomes induced macrophage M2 polarizationBy performing PCR,flow cytometry and ELISA,we revealed that hypoxic gliomaexosomes significantly induced macrophage M2 polarization.Moreover,hypoxic glioma exosome-treated macrophages could facilitate the proliferation,migration and invasion of glioma cells.For in vivo experiments,nude mice co-injected with glioma cells and hypoxic glioma exosomes-treated macrophages had shorter survival time and more aggressive gliomas.(2)MicroRNA-1246 was enriched in hypoxic glioma exosomes and glioma patients' cerebrospinal fluidThe microRNA sequencing analysis of glioma exosomes revealed that microRNA-1246 was enriched in hypoxic glioma exosomes.Moreover,microRNA-1246 was the most abundant microRNA in hypoxic glioma exosomes.PCR confirmed the microRNA sequencing results and demonstrated that microRNA-1246 could be delivered to recipient macrophages by hypoxic glioma exosomes,resulting in the increase of microRNA-1246 expression in macrophages.The microRNA sequencing analysis of glioma patients' cerebrospinal fluid determined that microRNA-1246 expression was correlated with glioma grades and patients' operation status.(3)MicroRNA-1246 induced macrophage M2 polarizationBy performing PCR,flow cytometry and ELISA,we revealed that microRNA-1246 significantly induced macrophage M2 polarization.Moreover,microRNA-1246-overexpressing macrophages could facilitate the proliferation,migration and invasion of glioma cells.For in vivo experiments,nude mice co-injected with glioma cells and microRNA-1246-overexpressing macrophages had shorter survival time and more aggressive gliomas.(4)Hypoxic glioma exosomal microRNA-1246 directly targeted TERF2IP to induce macrophage M2 polarizationBy performing mRNA sequencing analysis of macrophages,we found TERF2IP was down-regulated by microRNA-1246.Moreover,PCR and Western blot indicated TERF2IP expression was regulated by hypoxic glioma exosomes and microRNA-1246.Luciferase reporter assay confirmed microRNA-1246 could bind with TERP2IP 3'UTR.By performing PCR,flow cytometry and ELISA,we revealed that TERF2IP knockdown significantly induced macrophage M2 polarization.Moreover,TERF2IP-knockdown macrophages could facilitate the proliferation,migration and invasion of glioma cells.In addition,overexpression of TERF2IP attenuated the effect of microRNA-1246 on macrophage M2 polarization.(5)Hypoxic glioma exosomal microRNA-1246 induced macrophage M2 polarization by regulating STAT3 and NF-?B signaling pathwaysWestern blot results indicated that hypoxic glioma exosomes could influence the expression of proteins associated with STAT3 and NF-?B signaling pathways.Similarly,microRNA-1246 and TERF2IP could also regulate the expression of proteins associated with STAT3 and NF-?B signaling pathways.4.Conclusions(1)Hypoxic glioma exosomal microRNA-1246 induced macrophage M2 polarization by directly targeting TERF2IP via STAT3 and NF-?B signaling pathways.(2)MicroRNA-1246 was enriched in glioma patients' cerebrospinal fluid and had clinical relevance in glioma patients.MicroRNA-1246 could be a novel target for anti-glioma treatment.Part ?:The effect and mechanism of hypoxic glioma exosomes on normoxic glioma migration and invasion1.Objective(1)To determine the effect of hypoxic glioma exosomes on normoxic glioma migration and invasion.(2)By performing microRNA sequencing on glioma exosomes,to determine the microRNAs which are enriched in hypoxic glioma exosomes and may be associated with glioma migration and invasion(microRNA-1246 and microRNA-10b-5p).(3)To determine the effect of microRNA-1246 and microRNA-10b-5p on glioma migration and invasion and demonstrate the underlying mechanisms.(4)To determine the clinical relevance of microRNA-1246 and microRNA-10b-5p in glioma patients.2.Method(1)Isolation and identification of exosomesBy ultracentrifugation and precipitation methods,we isolated exosomes from supernatants of glioma cell lines.The exosomes were identified by transmission electron microscope(TEM),qNano analysis and Western blot.(2)Determination of the glioma migration and invasionThe methods to determine glioma migration and invasion abilities are as below:?Transwell assay was used to determine the migration and invasion of glioma cells.?3D glioma spheroid invasion assay was used to examine the invasion ability of glioma cells.(3)MicroRNA sequencing of glioma exosomesAfter exosome isolation,microRNA sequencing was performed to determine the expression pattern of microRNAs in normoxic and hypoxic glioma exosomes.(4)Detection of microRNA,mRNA and protein levelsSpecial treatments were used to extract the total RNA or proteins from the exosomes or treated cells,and PCR and Western blot were used to detect the expression levels of microRNAs,mRNAs and proteins.(5)Cell transfection and construction of microRNA-1246-expressing glioma cell lines and microRNA-10b-5p-expressing glioma cell linesMicroRNA-1246 mimics,mcroRNA-10b-5p mimics,FRK siRNAs,TFAP2A siRNAs,FRK-overexpression plasmids,TFAP2A overexpression plasmids,luciferase reporter plasmids containing mutant or wild-type FRK 3'UTR binding domain,luciferase reporter plasmids containing mutant or wild-type TFAP2A 3'UTR binding domain were used in cell transfection with Lipo3000.The purpose of cell transfections was to knockdown or overexpress target molecules to study the phenotype changes.The microRNA-1246 overexpression lentivirus were used to transfect the glioma cell lines to construct microRNA-1246-expressing glioma cell lines.The microRNA-10b-5p overexpression lentivirus were used to transfect the glioma cell lines to construct microRNA-10b-5p-expressing glioma cell lines.(6)Models of human GBM in nude mice by orthotopic transplantationLuciferase-labeled U87MG glioma cells with different treatment were injected into the brains of nude mice.Bioluminescence imaging was used to image the tumors and nude mice were followed up every day.Tumor samples from xenograft models were used for the further studies.3.Results(1)Hypoxic glioma exosomes promoted normoxic glioma migration and invasionBy performing Transwell assay and 3D spheroid invasion assay,we revealed that hypoxic glioma exosomes significantly promoted normoxic glioma cells migration and invasion.Moreover,hypoxic glioma exosome-treated normoxic glioma cells had higher expression of MMP2,MMP9,N-cadherin and Vimentin.For in vivo experiments,nude mice co-injected with glioma cells and hypoxic glioma exosomes had shorter survival time and more aggressive gliomas.(2)MicroRNA-1246 and microRNA-10b-5p were enriched in hypoxic glioma exosomesThe microRNA sequencing analysis of glioma exosomes revealed that microRNA-1246 and microRNA-10b-5p were enriched in hypoxic glioma exosomes.PCR confirmed the microRNA sequencing results and demonstrated that microRNA-1246 and microRNA-10b-5p could be delivered to recipient normoxic glioma cells by hypoxic glioma exosomes,resulting in the increase of microRNA-1246 and microRNA-10b-5p expression in normoxic glioma cells.By using CGGA database,we determined the clinical relevance of microRNA-1246 and microRNA-10b-5p in glioma patients.(3)MicroRNA-1246 and microRNA-10b-5p promoted glioma cell migration and invasionBy performing Transwell assay and 3D spheroid invasion assay,we revealed that microRNA-1246 and microRNA-10b-5p significantly promoted glioma cells migration and invasion.Moreover,microRNA-1246 and microRNA-10b-5p could increase the expression of MMP2,MMP9,N-cadherin and Vimentin in glioma cells.For in vivo experiments,nude mice injected with microRNA-1246-overexpressing glioma cells or microRNA-10b-5p-overexpressing glioma cells had shorter survival time and more aggressive gliomas.(4)MicroRNA-1246 directly targeted FRK and microRNA-10b-5p directly targeted TFAP2A to promote glioma cell migration and invasionWestern blot indicated FRK expression was regulated by microRNA-1246 and TFAP2A expression was influenced by microRNA-10b-5p.Luciferase reporter assay confirmed microRNA-1246 could bind with FRK 3 'UTR and microRNA-10b-5p could bind with TFAP2A 3'UTR.By performing Transwell assay,we revealed that FRK and TFAP2A knockdown significantly promoted glioma migration and invasion.Moreover,overexpression of FRK attenuated the effect of microRNA-1246 on glioma migration and invasion.And overexpression of TFAP2A inhibited the function of microRNA-10b-5p in glioma migration and invasion.4.Conclusions(1)Hypoxic glioma exosomes delivered microRNA-1246 and microRNA-10b-5p to normoxic glioma cells,therefore promoting glioma migration and invasion by directly targeting FRK and TFAP2A respectively.(2)MicroRNA-1246 and microRNA-10b-5p had clinical relevance in glioma patients and they could be novel targets for anti-glioma treatment.Part ?:The function of PLEKHG5 in gliomas1.Objective(1)To reveal the expression pattern of PLEKHG5 in glioma tissues.(2)To determine the clinical relevance of PLEKHG5 in glioma patients.(3)To demonstrate the function of PLEKHG5 on glioma cells.2.Method(1)Immunohistochemistry staining of clinical specimens61 cases of clinical glioma tissue and 6 cases of normal brain tissue were collected and used for the immunohistochemistry staining.(2)Bioinformatics analysis about PLEKHG5Analysis of genes associated with PLEKHG5 was performed by using the TCGA database.GO,KEGG and GSEA analysis determined the potential biological processes and signaling pathways which PLKEHG5 may be involved in.(3)Cell transfectionPLEKHG5 siRNA was used in cell transfection with Lipo3000.The purpose of cell transfection was to knockdown PLEKHG5 to study the phenotype changes.(4)Determination of the glioma migrationTranswell assay was used to determine the migration of glioma cells.(5)Detection of protein levelsSpecial treatments were used to extract the total proteins from the treated cells and Western blot was used to detect the expression levels of special proteins.3.Results(1)The expression of PLEKHG5 was related with glioma gradesThe results of immunohistochemistry staining indicated that compared to normal brain tissues,glioma tissues had a relatively higher expression level of PLEKHG5.The expression of PLEKHG5 was associated with glioma grades.Moreover,GBMs had higher ratio and stronger intensity of PLEKHG5 expression.(2)The expression pattern of PLEKHG5 was related with the glioma patients'prognosisBy analyzing the immunohistochemistry staining results and the prognosis of glioma patients we involved,we found higher expression levels of PLEKHG5 in gliomas indicated poorer patient survival.(3)Bioinformatics analysis revealed PLEKHG5 function in gliomasGO,KEGG and GSEA analysis determined that PLEKHG5 was involved in extracellular matrix disassembly,regulation of actin cytoskeleton and epithelial-mesenchymal transition and could regulate VEGF,PI3K and HIF-1 signaling pathways.(4)PLEKHG5 influence glioma migration and invasionBy performing migration assay,we demonstrated PLEKHG5 siRNA could inhibit glioma migration.In addition,Western blot results indicated that the expression of MMP2,MMP9 and mDia was regulated by PLEKHG5.4.Conclusions(1)The expression of PLEKHG5 was related with glioma grades and glioma patients' survival.PLEKHG5 may be a novel biomarker for glioma diagnosis and prognosis.(2)PLEKHG5 could facilitate glioma migration and invasion and may be a novel target for anti-glioma treatment. |
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