Effect And Molecular Mechanism Of Exosomes-derived From The Embryonic Stem Cells Inhibition Of Glioma

BACKGROUND AND OBJECTIVE:Glioma is the most common primary intracranial tumor,it is difficult to achieve satisfactory therapeutic effect at present,so it is urgent to develop new therapeutic methods.Previous studies have shown that stem cells,including mesenchymal stem cells and embryonic stem cells,are tumor prone and inhibit tumor growth.Mesenchymal stem cells(MSCs)are derived from early mesoderm,it is a kind of adult stem cells with self renewal and multi differentiation potential and usually be extracted from bone marrow,adipose tissue,umbilical cord tissue or umbilical cord blood.Studies have shown that the bone marrow derived MSCs injected via caudal vein can target to subcutaneous Kaposi sarcoma sites and inhibit the tumor growth,and MSCs can also migrate from the normal lateral brain tissue to the location of the lateral brain glioma and inhibit tumor growth.Embryonic stem cells(ESCs)belong to pluripotent stem cells and derive from blastocyst inner cell mass or early abortion fetal primordial gonad.It can limitlessly proliferate in vitro and has the characteristics of totipotent differentiation.And some studies have shown that ESCs microenvironment can inhibit the malignant phenotype of melanoma and breast cancer cells.Recent studies have shown that stem cells play a biological role mainly through the paracrine mechanism,and exosomes play a crucial role.Exosomes are a class of nanoscale vesicles,they can transport biological cells from their biological membranes to the target cells,such as proteins,lipids and nucleic acids,which play a "messenger" role between their source cells and target cells.Studies have shown that exosomes secreted by MSCs can inhibit liver cancer,ovarian cancer,breast cancer and bladder cancer growth,and exosomes secreted by ESCs(ESC-exos)can make the malignant phenotype of tumor to benign change,suggesting that they also have tumor suppressor role,but the role of ESC-exos in glioma have not been reported.Because the MSCs are often obtained from the invasive operation,the qulity of MSCs is affected by diabetes and other basic diseases and age,and amplification ability in vitro is limited.However,ESCs maintain the infinite proliferation of undifferentiated state in vitro,and can provide a sufficient source of exosomes,therefore ESCs are an ideal seed cells.Besides,exosomes have the advantages of low immunogenicity,stable biological activity,convenient storage and transportation.Hence,exosomes show good prospects for clinical transformation and application.In our previous study,we found that ESCs-derived exosomes(ESC-exos)has significant function in promoting the regeneration of damaged tissue,and they also have the ability to inhibit the growth of tumor cells.On the basis of previous studies,the inhibitory effect of ESC-exos on the growth of glioma was first determined through a series of experiments in vitro and in vitro.Then we analyzed the small RNA in ESCexos by small RNA sequencing technology,and we selected and evaluated the role of the key small RNA with high expression abundance to the growth of glioma.We evaluated the effect of the key small RNA on the proliferation and migration of glioma cells and vascular endothelial cells in vitro,and evaluated the effect of the key small RNA on the growth of glioma by in vivo experiments.Further,transcriptome sequencing technology was used to evaluate the effect of key small RNA on gene expression profiles of glioma cells,with a focus on tumor angiogenesis related genes to determine the molecular mechanism of ESC-exos inhibition of glioma,and this will provide a new strategy for the treatment of glioma.We also analyzed the effects of ESC-exos in mouse ischemic hind-limbs to clarify the role of ESC-exos on angiogenesis in the ischemic injury and tumor microenvironment,thus providing a theoretical basis for the application of ESC-exos in the treatment for glioma.METHODS:1.Extraction and identification of ESC-exos: ESCs H9 was cultured in a feeder free system,and ESC-exos were purified by ultrafiltration combined with ultracentrifugation and then identified by transmission electron microscopy,western blot and q Nano.2.Evaluation of the effect of ESC-exos on the growth of glioma: The effects of ESCexos on the biological behavior of cultured glioma and other tumor cells were evaluated in vitro experiments.By pathological techniques,the effects of ESC-exos on glioma growth were evaluated with nude mice bearing tumor as the animal model in vivo experiments.3.Analysis of molecular mechanism of ESC-exos inhibition of the growth of glioma: Small RNAs carried by ESC-exos were analyzed by throughput sequencing technology.The effects of the critical small RNA carried by ESC-exos on the biological behavior of cultured glioma cells and vascular endothelial cells were evaluated in vitro experiments.By pathological detection techniques,the effects of key small RNA carried by ESC-exos on glioma growth were evaluated by using nude mice bearing tumor in vivo experiments.4.Analysis of molecular mechanism of key small RNA carried by ESC-exos inhibition of the growth of glioma: The level of key small RNA in glioma cells was regulated by cell transfection,and the differentially expressed genes were detected by transcriptional sequencing and verified by real-time fluorescence quantitative PCR.The effects of conditioned medium from glioma cells transfected by key small RNA on the proliferation,migration and tube formation of vascular endothelial cells were verified in vitro experiments,and the protein concentration in the culture supernatant was measured by ELISA,so as to clarify the molecular mechanism of ts RNA carried by ESC-exos inhibition of glioma.5.Exploration of the effect of exosomes-derived from stem cells on angiogenesis in different tissue microenvironments: To evaluate the effects of ESC-exos,USC-exos and i MSC-exos on the repair of ischemic limbs and the angiogenesis in the injured tissues by behavioral test,laser Doppler blood flow detection and pathological techniques in a mouse hind-limb ischemic model,so as to clarify the different effect of ESC-exos on angiogenesis in ischemic injury and tumor microenvironments.RESULTS:1.ESC-exos can significantly inhibit the biological behavior of glioma cells:(1)Experiment results in vitro showed that ESC-exos could significantly inhibit the proliferation and migration of U87 and U251 glioma cells;(2)The in vivo experiment results show that ESC-exos can significantly inhibit the growth of U87 glioma,and the immunohistochemical staining results showed that ESC-exos could significantly inhibit the proliferation of cells and tumor angiogenesis.2.ESC-exos plays an antitumor effect by transmitting ts RNA-Val and ts RNA-Gly to the target cells:(1)Small RNA high throughput sequencing results indicate that ESCexos enriched a large number of ts RNA fragments derived from transport RNA,including high abundance of ts RNA-Val and ts RNA-Gly;(2)Immunofluorescence staining showed that U87,U251 and human microvascular endothelial cells HMEC-1 can uptake the Di I fluorescent dye labeled ESC-exos;(3)In vitro experiments showed that both ts RNA-Val and ts RNA-Gly could inhibit the proliferation and migration of U87,U251 and HMEC-1 cells;(4)In vivo experiments showed that ts RNA could significantly inhibit the volume and weight of glioma in vivo.Immunohistochemical staining showed that ts RNA could significantly inhibit cell proliferation and angiogenesis in tumor tissues;(5)WGCNA analysis of RNA-seq results showed that ts RNA down regulated the expression of proliferation related genes in U87 cells,such as cell cycle related genes,DNA damage repair related genes,etc.Besides,GSEA analysis showed that there was a significant correlation between ts RNA and angiogenesis;(6)The proliferation,migration and tubule formation of vascular endothelial HMEC-1 were significantly inhibited by the cell culture supernatant of U87 glioma cells,which was regulated by the key small RNA;(7)q PCR results confirmed that ts RNA altered the gene expression profile of angiogenesis related secretory protein in U87 cells,that is,the expression of pro-angiogenic genes ALCAM,CTGF,HBEGF,ID1 and VEGFC were down regulated,and the anti-angiogenic gene THBS2 was upregulated;and the results of the ELISA experiment showed that the protein concentration also correspondingly changed.3.ESC-exos can repair the ischemic limbs via promoting angiogenesis:(1)The results of laser Doppler blood flow detection showed that ESC-exos,USC-exos and i MSCexos could promote the blood flow perfusion of the ischemic limbs;(2)The results of behavioral assessment showed that all three kinds of stem cell-derived exosomes could reduce the injury and necrosis score of ischemic limbs;(3)The results of tissue immunofluorescence staining showed that all three stem cell-derived exosomes could promote angiogenesis in ischemic limbs,and ESC-exos showed a more significant angiogenesis promoting effect.CONCLUTIONS:1.ESC-exos can significantly inhibit the growth of glioma.2.ESC-exos inhibits the growth of glioma by inhibiting tumor angiogenesis through regulating the expression of angiogenesis related genes in glioma cells by transferring tsRNA.3.ESC-exos show a "double" effect on angiogenesis : ESC-exos can promote angiogenesis in ischemic injury tissue,but they play an opposite effect in tumor,which may be influenced by tissue microenvironment,and its exact mechanism needs further study.4.ESC-exos have broad clinical application prospects because ESC-exos have many advantages,such as abundant sources,convenient for industrialization,storage and transportation,and easy to pass blood-brain barrier and so on.And the discovery of the inhibitory effect of ESC-exos on glioma will provide a new technical strategy to overcome the treatment predicament of glioma.