The Functional And Mechanism Studies Of Exosomes Derived From Macrophages And Mesenchymal Stem Cells In Occurrence And Development Of Glioma

BackgroundGlioma is the most common primary malignant brain tumor in adults.Glioblastoma(WHO grade ?)is a type with the highest malignancy.The median survival time for glioblastoma patients is no more than 16 months.Despite the application of various adjuvant treatments including chemotherapy,radiotherapy,and antiangiogenic therapy,the overall prognosis of the patient has not been significantly improved for many years.Mesenchymal transition and vasculogenic mimicry(VM)formation are two significant signs relative to poor prognosis,and are important factors triggering the occurrence and development of gliomas.Mesenchymal transition enables glioma cells to gain a more malignant phenotype,while VM formation provides a malignancy-promoting histological structure for gliomas.In addition,both of them are related to the treatment resistance of glioma,making it more difficult to interfere with the occurrence and development of the desease.Exploring the specific mechanism of glioma mesenchymal transition and the formation of vasculogenic mimicry,or further finding the corresponding solutions,will provide a theoretical basis for slowing down the occurrence and development of gliomas and improving the prognosis for glioma patients.The tumor immune microenvironment(TIME)contains a variety of immunosuppressive cellular components,such as:tumor-associated macrophage(TAM),myeloid-derived suppressor cell(MDSC),regulatory T cell(Treg),etc.,among which tumor-associated macrophages are the most important immunosuppressive cells in gliomas.Our previous studies have confirmed that tumor-associated macrophages promote the malignant behaviors including proliferation and invasion in gliomas.In addition,studies have shown that tumor-associated macrophages are adjacent to mesenchymal glioma cells in glioma tissue,suggesting that tumor-associated macrophage is probably a key factor for mesenchymal transition.Confirming this hypothesis will explain the reasons for mesenchymal transition and provide theoretical support for inhibiting the occurrence and development of gliomas.Exosomes are 50-100 nanometer vesicles secreted by cells,and they are important media for the communication process between cells.For example,exosomes are delivery media for microRNAs between tumor cells and tumor-associated immune cells,enabling the exchange of materials and information between the cells.Exploring the effect of tumor-associated macrophage exosomes on gliomas may help to discover the specific mechanism of mesenchymal transition of gliomas.In addition,considering the material delivery ability of exosomes,modified exosomes can deliver tumor suppressive molecules or anti-tumor drugs to tumor cells,thereby inhibiting tumor progression.Finding safe and handy exosomal donor cells will be the key to modifying exosomes for glioma inhibition.Mesenchymal stem cells(MSC)are a type of pluripotent stem cells derived from the mesoderm,with strong self-renewal ability and multi-directional differentiation potential.Since mesenchymal stem cells have a wide range of sources,are easy to cultivate,and can secrete a large number of exosomes,they have become an important source of anti-tumor exosomes.Aiming at the key steps in the occurrence and development of gliomas,the modification and utilization of mesenchymal stem cell-derived exosomes is prospective for inhibiting gliomas.Based on the gene signature of gliomas,gliomas can be divided into three molecular subtypes:proneural,classical,and mesenchymal.Among them,the proneural glioma has the lowest degree of malignancy and the best prognosis;while the mesenchymal glioma has the strongest degree of malignancy and the worst prognosis.The proneural-to-mesenchymal transition(PMT)refers to the transformation of the molecular phenotype of glioma from the proneural to mesenchymal during the occurrence,development and recurrence of gliomas.This process leads to a significant increase in the malignancy of gliomas,which further accelerates the occurrence and development of gliomas.Radiotherapy is one of the important adjuvant treatments for glioma.The resistance of glioma to radiotherapy is closely related to the transition process between its molecular subtypes.Studies have confirmed that the proneural-to-mesenchymal transition caused a sharp increase in the resistance of glioma to radiotherapy.Therefore,exploring the specific mechanism of proneural-to-mesenchymal transition will be of great significance for slowing down the occurrence and development of gliomas and reducing the resistance of gliomas to radiotherapy.Vasculogenic mimicry(VM)is an abnormal tubular structure in glioma tissues,which can provide sufficient blood supply for tumor tissues in the same pattern as normal blood vessels,and promote the occurrence and development of tumors.As a commonly used antiangiogenic therapy drug for tumors,the main mechanism of bevacizumab is to block the vascular endothelial growth factor(VEGF),thereby targeting tumor vascular endothelial cells and inhibiting the formation of tumor blood vessels.However,unlike general blood vessels,the formation of vasculogenic mimicry does not rely on vascular endothelial cells.Therefore,vasculogenic mimicry exhibits strong resistance to antiangiogenic therapy.There is even a compensatory increase of VM after antiangiogenic therapy to maintain the blood supply of the tumor.In view of the above characteristics,vasculogenic mimicry is an important factor in the resistance to antiangiogenic therapy,which affects the occurrence,development and therapeutic efficacy of gliomas.Exploring the mechanism of the formation of vasculogenic mimicry and looking for corresponding treatments will be expected to inhibit the occurrence and development of gliomas and improve the prognosis of patients with gliomas.The purpose of this study is to explore the driving factors for the occurrence and development of gliomas,and the limitations of radiotherapy and antiangiogenic treatments for gliomas are set as the starting point.Based on "proneural-to-mesenchymal transition" and "vasculogenic mimicry",we studied the key role of exosomes in the proneural-to-mesenchymal transition,and the potential application value of exosomes in the anti-vasculogenic mimicry treatment of gliomas,respectively.First,we used M2 macrophages as the model for tumor-associated macrophages,and confirmed that their exosomes can promote the proneural-to-mesenchymal transition and the radiotherapy resistance of glioma stem cells.In addition,we confirmed miR-27a-3p,miR-22-3p and miR-221-3p mediate this process through a series of downstream molecular pathways.Secondly,we found the inhibitory effect of miR-29a-3p on glioma vasculogenic mimicry.Human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p could inhibit the formation of vasculogenic mimicry in vivo and in vitro.On the one hand,this doctoral thesis explored the mechanism of proneural-to-mesenchymal transition of glioma,and discovered an important inhibitory molecule of glioma vasculogenic mimicry;on the other hand,it provides potential for solving the radiotherapy resistance of glioma,and provide novel ideas for reducing vasculogenic mimicry.Part ?.The effect and mechanism of M2 macrophage-derived exosomes on proneural-to-mesenchymal transition in glioma1.Objectives(1)Explore the differences in miR expression profiles between M2 macrophage-derived exosomes and M0 macrophage-derived exosomes.(2)Investigate the effects of M2 macrophage-derived exosomes on glioma stem cells inducing proneural-to-mesenchymal transition and radiotherapy resistance.(3)Identify the molecular mechanisms through which M2 macrophage-derived exosomes promote the proneural-to-mesenchymal transition and radiotherapy resistance in glioma stem cells.2.Materials and Methods(1)Culture of M2 macrophages and extraction,identification of exosomesFirstly,the peripheral blood mononuclear cells of healthy volunteers were collected,and the CD 14-positive monocytes were obtained by magnetic cell sorting method.The CD 14-positive cells were artificially stimulated to differentiate into M0 and M2 macrophages.Secondly,the MO and M2 macrophage-derived exosomes were extracted by ultracentrifugation,and they were verified by transmission electron microscope.In addition,Nanosight and Western Blot are used to analyze the concentration,diameter and molecular markers of exosomes.(2)The uptake assay of macrophage-derived exosomes by glioma stem cellsM0 and M2 macrophage-derived exosomes were labeled with PKH-67,and were co-cultured with glioma stem cells in vitro.Confocal microscope was used to observe the uptake of macrophage-derived exosomes by glioma stem cells.(3)Verification of M2 macrophage-derived exosomes inducing proneural-to-mesenchymal transition and radiotherapy resistance in vitro and in vivoM0 and M2 macrophage-derived exosomes were used to stimulate proneural glioma stem cells in vitro,and the self-renewal ability and proliferation ability of glioma stem cells were detected by tumorsphere formation assay,limiting dilution assay and EdU cell proliferation assay.Flow cytometry was used to detect changes in the expression of the mesenchymal marker CD44;Western Blot was used to detect changes in the protein levels of proneural and mesenchymal markers.The glioma stem cells stimulated by macrophage-derived exosomes were treated with 6 Gy dose of radiotherapy,and the apoptosis and cell cycle changes were detected by flow cytometry.Glioma stem cells pre-stimulated in vitro with macrophage-derived exosomes were inoculated to nude mice for in situ tumor formation,and the tumor-bearing mice were treated with radiotherapy.The tumor size changes were observed by ex vivo bioluminescent imaging,and immunohistochemistry staining was used for detecting the expression of mesenchymal glioma markers.The resistance of nude mice in different treatment groups to radiotherapy was judged by Tunel staining technique.(4)miR sequencing and key miR screening for M0 and M2 macrophage-derived exosomesUsing the Illumina HiSeq 2500 sequencing platform,miR sequencing was performed for M0 and M2 macrophage-derived exosomes,and the top 15 most-expressed miRs that were up-regulated in M2 macrophage-derived exosomes were screened out.The above 15 miRs were overexpressed in glioma stem cells,and their effects on the expression of CD44 were detected by flow cytometry.Finding the miR that can up-regulate the expression of CD44 and further verifying their effects on the self-renewal and proliferation of glioma stem cells,and confirming their effects on the resistance to radiotherapy in glioma stem cells.(5)Prediction and verification of downstream targets of miR-27a-3p,miR-22-3p and miR-221-3pUse miR downstream target prediction sites TargetScan and miRDB to predict the downstream targets of miR-27a-3p,miR-22-3p and miR-221-3p,and compare the predicted common downstream targets of the three miRs with a known proneural glioma gene set.The intersection of predicted downstream targets and proneural glioma gene set denoted the common targets of miR-27a-3p,miR-22-3p and miR-221-3p.The direct binding of miR-27a-3p,miR-22-3p and miR-221-3p to downstream targets was verified by luciferase reporter gene assay and Western Blot.After the knocking down the downstream target in glioma stem cells,perform flow cytometry to detect changes in the expression of CD44 molecules.The glioma stem cells knocked down the target molecule were treated with 6 Gy radiotherapy,and their apoptosis and cell cycle changes were detected by flow cytometry.(6)Verification of the effects of miR-27a-3p,miR-22-3p,miR-221-3p and their downstream targets on proneural-to-mesenchymal transition and radiotherapy resistance in vitroGlioma stem cells stably overexpressing miR-27a-3p,miR-22-3p and miR-221-3p and stably knocking down their common downstream targets were injected into the brain of nude mice.The tumor-bearing mice were treated with radiotherapy,and ex vivo bioluminescent imaging technology was used to observe tumor size changes.Immunohistochemistry staining was used for detecting tumor mesenchymal glioma markers,and Tunel staining was used to judge the resistance to radiotherapy for nude mice in different treatment groups.(7)Research on downstream molecular pathways of miR-27a-3p,miR-22-3p,miR-221-3p and their targetsUse the gene set enrichment analysis(GSEA)method to predict the downstream pathway,and use the Western Blot method to verify the protein expression of the key factors in these pathways.3.Results(1)M2 macrophage-derived exosomes can be taken up by glioma stem cells,and promote proneural-to-mesenchymal transition and radiotherapy resistance in glioma stem cells in vitroThe exosomes extracted by ultracentrifugation are eumorphism in shape,about 100 nanometers in diameter,and express the exosomal markers TSG101 and CD9.Confocal microscopy observations confirmed that macrophage-derived exosomes can be taken up by glioma stem cells,which are shown as punctate structures with PKH-67 staining found in the cytoplasm of glioma stem cells.By co-culture macrophage-derived exosomes and glioma stem cells,we found that M2 macrophage-derived exosomes can promote the self-renewal ability of glioma stem cells(tumorsphere formation assay and limiting dilution assay)and proliferation ability(EdU staining).The results of flow cytometry showed that M2 macrophage-derived exosomes can promote the increase in the expression of CD44,a mesenchymal marker of glioma stem cells;at the same time,Western Blot results confirmed that M2 macrophage-derived exosomes have an up-regulatory effect on mesenchymal markers YKL40 and CD44,and a down-regulatory effect on proneural marker SOX2.Radiotherapy was performed on glioma stem cells stimulated by macrophage-derived exosomes,and it was found that glioma stem cells stimulated by M2 macrophage-derived exosomes showed lower levels of apoptosis and less cells blocked in G2/M phase.(2)M2 macrophage-derived exosomes promote proneural-to-mesenchymal transition and radiotherapy resistance in vivoGlioma stem cells pre-stimulated in vitro with macrophage-derived exosomes were injected to nude mice,and then the nude mice were divided into radiotherapy group and non-radiotherapy group.Ex vivo bioluminescent imaging results showed that the growth rate of glioma stem cells treated with M2 macrophage-derived exosomes in mice was significantly faster than that of control group or M0 macrophage-derived exosomes group.Immunohistochemical staining revealed that M2 macrophage-derived exosomes up-regulated the expression of mesenchymal marker YKL40 in transplanted tumors.Tunel staining was performed on the transplanted tumor tissue of nude mice after radiotherapy.It was found that the transplanted tumor treated with M2 macrophage-derived exosomes had fewer Tunel-positive cells after radiotherapy,suggesting its resistance to radiotherapy-induced apoptosis.(3)The up-regulated miR-27a-3p,miR-22-3p and miR-221-3p in M2 macrophage-derived exosomes promoted the proneural-to-mesenchymal transition in glioma stem cellsUsing the Illumina HiSeq 2500 sequencing platform,miR sequencing were performed on M0 and M2 macrophage-derived exosomes,and the 15 miRs that were up-regulated and most expressed in M2 macrophage-derived exosomes were transfected into glioma stem cells.The results show that miR-27a-3p,miR-22-3p and miR-221-3p can significantly promote the expression of mesenchymal markers of glioma stem cells,and at the same time enhance the self-renewal and proliferation ability.Knockdown of miR-27a-3p,miR-22-3p and miR-221-3p in M2 macrophage-derived exosomes showed that their promoting effect on proneural-to-mesenchymal transition is significantly offset.(4)miR-27a-3p,miR-22-3p and miR-221-3p promote the proneural-to-mesenchymal transition.of glioma stem cells by targeting CHD7Use miR downstream target prediction sites TargetScan and miRDB to predict the downstream targets of miR-27a-3p,miR-22-3p and miR-221-3p,and compare the predicted common downstream targets of the three miRs with a known proneural glioma gene set.The intersection of predicted downstream targets and proneural glioma gene set revealed that CHD7 may be a potential downstream target of the three miRs.The results of luciferase reporter gene assay and Western Blot verified the direct targeting of miR-27a-3p,miR-22-3p and miR-221-3p to CHD7.Knockdown of CHD7 molecules in glioma stem cells induced an increase in the expression of mesenchymal markers,confirming that it serves as a downstream target of miR-27a-3p,miR-22-3p and miR-221-3p,promoting the proneural-to-mesenchymal transition of glioma stem cells.(5)miR-27a-3p,miR-22-3p and miR-221-3p mediate the induction of M2 macrophage-derived exosomes on glioma stem cell radiotherapy resistance by targeting CHD7Glioma stem cells were transfected with miR-27a-3p,miR-22-3p and miR-221-3p,and a single dose of 6 Gy radiotherapy was administrated.It was found that miR-27a-3p,miR-22-3p and miR-221-3p overexpression significantly inhibit radiotherapy-induced glioma stem cell apoptosis and G2/M phase arrest.At the same time,knockdown of miR-27a-3p,miR-22-3p and miR-221-3p can promote radiotherapy-induced apoptosis and G2/M phase arrest.In addition,knockdown of CHD7 in glioma stem cells inhibited the effeect of radiotherapy.(6)Overexpression of miR-27a-3p,miR-22-3p and miR-221-3p or knockdown of CHD7 promotes the proneural-to-mesenchymal transition and radiotherapy resistance in vivoGlioma stem cells overexpressing miR-27a-3p,miR-22-3p and miR-221-3p or knockdown CHD7 were innoculated into brain of nude mice,and the nude mice were divided into radiotherapy group and non-radiotherapy group.Radiotherapy was performed on nude mice in the radiotherapy group.The results of ex vivo bioluminescent imaging showed that glioma stem cells overexpressing miR-27a-3p,miR-22-3p and miR-221-3p or knocking down CHD7 grew faster in the brain of mice with or without radiotherapy.Immunohistochemical staining showed that the overexpression of miR-27a-3p,miR-22-3p and miR-221-3p or the knockdown of CHD7 up-regulated the expression of the mesenchymal marker YKL40 in transplanted tumors.Tunel staining was performed on the transplanted tumor tissue of nude mice after radiotherapy,and it was found that transplanted tumors overexpressing miR-27a-3p,miR-22-3p and miR-221-3p or knocking down CHD7 had fewer Tunel-positive cells after radiotherapy,suggesting its resistance to radiotherapy.(7)miR-27a-3p,miR-22-3p and miR-221-3p in M2 macrophage-derived exosomes promote the proneural-to-mesenchymal transition of glioma stem cells through the CHD7/RelB/P50 and CHD7/p-STAT3 pathwaysGene set enrichment analysis(GSEA)was used to predict the downstream pathways related to CHD7,and it was found that the NF?B pathway and IL-6/STAT3 pathway are potentially related to CHD7.The results of Western blot partially verified the prediction,that is,the non-classical NF?B pathway CHD7/RelB/P50 and CHD7/p-STAT3 pathways jointly mediate the proneural-to-mesenchymal transition of glioma stem cells.4.Conclusion(1)M2 macrophage-derived exosomes can induce the proneural-to-mesenchymal transition of glioma stem cells and promote the resistance to radiotherapy.(2)miR-27a-3p,miR-22-3p and miR-221-3p enriched in M2 macrophage-derived exosomes are the key molecules for proneural-to-mesenchymal transition of glioma stem cells.(3)CHD7 is an important molecule for the maintenance of proneural phenotype in glioma stem cells.CHD7 regulates proneural-to-mesenchymal transition through the CHD7-RelB/P50 pathway and the CHD7-STAT3 pathway.(4)The increased expression of miR-27a-3p,miR-22-3p,miR-221-3p or the decreased expression of CHD7 are related to the poor prognosis of glioma patients.miR-27a-3p,miR-22-3p,miR-221-3p and CHD7 can be used as biomarkers for glioma diagnosis and radiotherapy efficiency evaluation.Part ?.The effect and mechanism of exosomes derived from mesenchymal stem cells on vasculogenic mimicry in glioma1.Objectives(1)Explore the key miRs regulate the formation and development of vasculogenic mimicry in glioma.(2)Aiming at the mechanism of vasculogenic mimicry formation,explore the treatment based on human mesenchymal stem cells to inhibit vasculogenic mimicry.2.Materials and Methods(1)Comparison of miR-29a-3p expression between normal brain tissue and glioma tissue or between normal human astrocyte and glioma cell lineFirst,perform statistical analysis on the expression of miR-29a-3p in normal brain tissues and different subtypes of gliomas in the TCGA database;then use qPCR to detect the expression level of miR-29a-3p in normal human astrocyte cell line and human malignant glioma cell lines U87 and A172.(2)Experiments of miR-29a-3p in inhibiting glioma migration ability and vasculogenic mimicry formation ability in vitroOverexpression and knockdown of miR-29a-3p were performed on the malignant glioma cell lines U87 and A172.The transwell migration experiment was used to detect the migration ability of glioma cells.The matrigel matrix was planked on the bottom of cell culture plate to provide a condition for vasculogenic mimicry formation.Then observe and detect the vasculogenic mimicry of glioma cells.In addition,Western Blot was used to detect the expression changes of migration-and vasculogenic mimicry-related proteins after overexpression and knockdown of miR-29a-3p.(3)Prediction and verification of downstream targets of miR-29a-3pUse miR downstream target prediction websites TargetScan and miRDB to predict miR-29a-3p downstream targets.Western Blot was used to verify the inhibitory effect of miR-29a-3p on protein expression of downstream target,and the luciferase reporter gene assay was used to confirm the direct binding effect of miR-29a-3p to downstream targets.Rescue experiment was performed.Overexpressing downstream target molecules and miR-29a-3p at the same time,then Western Blot was performed to detect migration-and vasculogenic mimicry-related proteins,transwell assay and vasculogenic mimicry formation assay were performed to examine the migration and vasculogenic mimicry formation abilities.(4)Extraction and identification of exosomes from human mesenchymal stem cells overexpressing miR-29a-3pFirst,lentivirus was used to transfect human mesenchymal stem cells to stably overexpress miR-29a-3p,and the human mesenchymal stem cells transfected with a nonsense RNA sequence were used as a negative control.Subsequently,the exosomes of mesenchymal stem cells were extracted by ultracentrifugation and verified by transmission electron microscopy at the morphological level.In addition,Nanosight and Western Blot were used to analyze the number,diameter and molecular markers of exosomes.Afterwards,the.expression level of miR-29a-3p in exosomes was detected by qPCR to confirm that miR-29a-3p can be secreted into exosomes by human mesenchymal stem cells overexpressing miR-29a-3p.(5)Verification of the effect of human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p on the migration and vasculogenic mimicry of gliomaThe malignant glioma cell lines U87 and A172 were co-cultured with human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p,and qPCR was used to detect the change of miR-29a-3p expression in glioma cells.Transwell migration assay was used to detect the migration ability of glioma cells.Detection of vasculogenic mimicry of glioma cells was conducted in a cell culture plate covered with matrigel matrix at the bottom.(6)In vivo experiments verifying the inhibitory effects of miR-29a-3p and human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p on the growth of gliomas and the formation of vasculogenic mimicryU87 glioma cells stably overexpressing or knocking down miR-29a-3p were intracranial inoculated into nude mice.At the same time,two groups of nude mice were transplanted with untransfected U87 glioma cells,and the negative control exosomes or human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p were given via the tail vein,respectively.Ex vivo bioluminescent imaging was used to observe tumor size changes,immunohistochemistry staining was used to observe the vasculogenic mimicry structure(PAS+/CD34-),and H&E staining was used to observe the invasion of glioma.3.Results(1)The expression of miR-29a-3p is significantly down-regulated in gliomasAnalysis of the expression of miR-29a-3p in normal brain tissue and different subtypes of glioma tissue in the TCGA database showed that the expression of miR-29a-3p in each subtype of glioma tissue was lower than that in normal brain tissue.Secondly,the qPCR results showed that the expression of miR-29a-3p in the human malignant glioma cell lines U87 and A172 were significantly lower than that of the normal human astrocyte cell line NHA.(2)miR-29a-3p inhibits glioma migration and vasculogenic mimicry formation in vitroTranswell cell migration assay was performed on the malignant glioma cell lines U87 and A172 overexpressing or knocking down miR-29a-3p.The results showed that miR-29a-3p can significantly inhibit the migration ability of glioma cell lines.At the same time,the malignant glioma cell line overexpressing or knocking down miR-29a-3p was inoculated on a cell culture plate covered with matrigel matrix at the bottom,and the vasculogenic mimicry of the glioma cells was observed.The results confirmed that miR-29a-3p inhibits the formation of vasculogenic mimicry.In addition,the results of Western Blot confirmed that miR-29a-3p can down-regulate the expression of proteins related to invasion,migration and vasculogenic mimicry.(3)ROBO1 is the downstream target of miR-29a-3p in regulating glioma migration and vasculogenic mimicryUsing miR downstream target prediction websites TargetScan and miRDB to predict the downstream targets of miR-29a-3p,we found that ROBO1 is a potential downstream target of miR-29a-3p.Western Blot results showed that miR-29a-3p can inhibit the expression of ROBO1,and the molecular changes related to invasion,migration and vasculogenic mimicry were decreased after ROBO1 were knocked down.The luciferase reporter gene experiment confirmed the direct binding effect of miR-29a-3p to ROBO1,Subsequently,we overexpressed ROBO1 in gliomas,and the results confirmed the promoting effect of ROBO1 on glioma migration and vasculogenic mimicry.However,overexpressing ROBO1 and miR-29a-3p at the same time can hinder ROBO1's promotion of migration and vasculogenic mimicry formation,indicating ROBO1 as a downstream molecule of miR-29a-3p.(4)miR-29a-3p is overexpressed in human mesenchymal stem cells,and the expression of miR-29a-3p in the secreted exosomes is also up-regulatedTransfecting human mesenchymal stem cells with miR-29a-3p or nonsense sequence as negative control,then the exosomes were extracted by ultracentrifugation.The nanoparticles maintained typical exosomal structures on the transmission electron microscopy photo.Nanosight assay confirmed that the nanoparticles were mostly 100 nanometers in diameter.Western Blot detection confirmed that the nanoparticles expressed exosomal markers TSG101 and CD9.miR-29a-3p expression in exosomes was detected by qPCR.The results confirmed that the expression of miR-29a-3p in exosomes secreted by human mesenchymal stem cells overexpressing miR-29a-3p was also up-regulated.(5)Human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p have a significant inhibitory effect on glioma migration and vasculogenic mimicryAfter stimulating the glioma cell lines U87 and A172 with negative control exosomes or human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p,qPCR results confirmed that the expression of miR-29a-3p in glioma cells was significantly up-regulated.At the same time,glioma cells stimulated by human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p showed reduced migration ability and vasculogenic mimicry formation ability.In addition,Western Blot results also confirmed that human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p can inhibit proteins related to invasion,migration and vasculogenic mimicry.(6)miR-29a-3p and human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p can inhibit the growth of glioma and reduced the formation of vasculogenic mimicry in vivoThe results of in vivo study showed that overexpression of miR-29a-3p or stimulation with human mesenchymal stem cell-derived exosomes overexpressing miR-29a-3p can reduce the size of transplanted tumors in nude mice.In addition,the results of immunohistochemical staining showed that miR-29a-3p overexpression or miR-29a-3p overexpressing exosome stimulation decreased the number of vasculogenic mimicry in the tumor tissues.The results of H&E staining suggested a reduced invasion ability after miR-29a-3p overexpression or miR-29a-3p overexpressing exosome stimulation.4.Conclusion(1)miR-29a-3p inhibits the formation of vasculogenic mimicry and cell migration in glioma.The expression level of miR-29a-3p is decreased in glioma.(2)The inhibitory effect of miR-29a-3p on the formation of vasculogenic mimicry and cell migration is mediated by directly targeting ROBO1.(3)Human mesenchymal stem cell-derived exosomes with high expression of miR-29a-3p can inhibit the formation of vasculogenic mimicry and cell migration in vitro and in vivo.