Effects And Mechanisms Of Exosomal MicroRNA-155-5p From Tumor Stem-like Cells On Invasiveness Of Glioma

Purpose: Glioma is the primary brain tumor with the highest incidence and has a poor clinical prognosis.Despite significant progress made in treatment,the 5-year survival rate for glioma patients remains <10%.Recently,more studies have indicated that exosomes are able to exert various biological functions via horizontally transferring many intercellular cargos,which also contribute to the formation of tumor microenvironment and participate in tumor progression,including malignant glioma.In our research,we detected the expression profile of microRNA in glioma stem-like cells-derived exosomes and identified the miR-155-5p was significantly up-regulated.We also elucidated its function and potential mechanism in glioma.Methods: Glioma stem-like cells（GSCs）were isolated from U87 cells by neural sphere culture methods and identified with immunofluorescence.GSCs-derived exosomes were isolated from the serum-free GSCs culture medium.Deep sequencing technology was used to explore the microRNA expression profile in GSCs-derived exosomes compared with exosomes released from human normal glia cells（HEB）.The result was identified by Real-time quantitative PCR（RT-q PCR）in exosomes from GSCs and HEB.The expression level of microRNA was modified by specific microRNA inhibitor in U87 and U251 cell lines.Wound healing and transwell experiments were performed to detect the aggressiveness of glioma cells.Bioinformatic tools,luciferase report and rescue experiments were used to elucidated the downstream target of microRNA and its potential mechanism.Results: The isolated glioma spheres were suspended in the neural culture medium.The surface marker of tumor stem cells（CD133）and neural cells（Nestin）were identified by immunofluorescence staining in glioma spheres.Wound healing and Transwell experiments indicated that GSCs-derived exosomes could enhance the aggressiveness of glioma cells.The result of deep sequencing showed that microRNA-155-5p was significantly up-regulated in GSCs-derived exosomes compared to HEB-derived exosomes.MiR-155-5p in plasma from glioma patients was also proved to be upregulated,which was also associated with tumor grading.Specific inhibition of miR-155-5p were able to attenuate the invasiveness and progression of glioma in vivo and in vitro.Furthermore,bioinformatic tools and luciferase report demonstrated that miR-155-5p directly targeted acetyl-Co A thioesterase 12（ACOT12）which was proved to be a tumor suppressor in glioma by immunohistochemistry and RT-q PCR.In addition,epithelial mesenchymal transition was also promoted by both GSCs-derived exosomes and overexpression of miR-155-5p.Therefore,up-regulated exosomal miR-155-5p from GSCs enhances invasiveness of glioma cells by promoting epithelial-mesenchymal transition（EMT）via targeting ACOT12.Conclusion:（1）GSCs-derived exosomes can promote the invasion and migration of glioma cells,which can be restrained by exosome secretion inhibitor.It indicated that the effect of glioma stem-like cells on surrounding tumor microenvironment was mediated by exosomes.（2）miR-155-5p was significantly up-regulated both in GSCs-derived exosomes and in plasma exosomes from glioma patients.Inhibition of miR-155-5p could suppress the development of glioma,which demonstrated that miR-155-5p played a vital role in glioma progression.（3）miR-155-5p was able to directly target ACOT12 and facilitate the epithelial mesenchymal transition,which promoted the progression of glioma.These findings indicated that exosomal mi R-155-5p could be the diagnostic and therapeutic target for glioma.