| Background and objectiveGBM is a tumor of the central nervous system.It is the malignant tumor with the highest incidence,and its main clinical features are rapid progress,strong aggressiveness and high recurrence rate.Patients with GBM have an average survival of just 10-15 months.Surgical treatment alone cannot cure the vast majority of GBM patients.Postoperative combined chemoradiotherapy can prolong the survival time of some patients,however,due to its strong invasiveness and recurrence,the actual therapeutic effect is still not ideal.The pathogenesis of GBM is not clear,and the molecular mechanism of its occurrence and development is not clear,so it is difficult to take targeted and effective treatment for GBM.Therefore,further and deeper studies on the molecular mechanism of glioblastoma development may bring breakthroughs in the treatment of GBM in the future.In recent years,more and more researchers have paid attention to the role of miRNA in tumor genesis and development.Researchers have found that miRNAs exhibit different characteristics and expression patterns in many different types of tumors,including GBM.Recently,researchers have focused on the role of miRNAs in GBM microenvironment communication,suggesting that miRNAs communicate and communicate in the tumor microenvironment mainly through the release and uptake of extracellular vesicles by tumor cells.Previous clinical studies in our center have found that down-regulation of miR-375 is associated with the progression of high-grade glioma and leads to a significant decrease in overall survival of glioma patients.However,the specific role of exosomes-delivered miR-375 in GBM remains unclear.This study started with the analysis of miRNA differentially expressed in GBM derived exosomes,focusing on the mechanism of miR-375 in GBM progression,to explore the expression level of miR-375 in GBM and its exosomes,to investigate the biological role of miR-375 in GBM,to clarify the intercellular transmission mode of miR-375,to try to treat GBM with miR-375 via exosomes,to explore the potential application prospect of exosome-delivered miR-375 as a new therapeutic approach for GBM in the future,and to provide new possibilities for molecular diagnosis and treatment of GBM.Methods1.Extraction and purification of exosomes.Exosomes were extracted and purified from cell supernatant and tissues by ultracentrifugation and density gradient centrifugation was used to purify the exosomes.2.Identification of exosomes.Western Blot,particle size analysis and transmission electron microscopy were used to detect whether the extracted exosomes complied with the accepted definitions of exosomes.3.Screening of differentially expressed miRNAs in exosomes from GBM tissues and paratumoral tissues.Ultracentrifugation and density gradient centrifugation was used to extract and purify exosomes from GBM tissues and paratumoral tissues,and miRNA sequencing technology was used to screen differentially expressed miRNAs.4.The detection of miR-375 expression level in GBM cell lines and their exosomes.A172,U87,U251,U373 and NHA cell lines were used for experiments.Exosomes from the above five cell lines were extracted and purified by ultracentrifugation and density gradient centrifugation.Mi R-375 levels in the above five cell lines and their exosomes were detected by q RT-PCR.5.Proliferation ability was detected by CCK-8 cell proliferation assay.6.Migration ability was detected by scratch healing assay.7.Invasion ability was detected by transwell invasion assay.8.To explore the biological role of miR-375 in vivo.miR-375 mimic and mimic NC lentivirus were used to infect U251 cells to establish stable cell lines.The model of in situ intracranial tumor formation in nude mice was established by stereotactic injection technique.Fluorescence imaging technique was used to detect brain fluorescence intensity.9.To explore the transfer method of miR-375 among tumor cells.U251 cells were infected with miR-375 mimic and mimic NC lentivirus to establish stable cell lines.Two stable cell lines were co-cultured with the receptor U251,and the miR-375 level in the receptor U251 cells was detected by q RT-PCR.Exosomes derived from two stable cell lines were extracted and purified by ultracentrifugation and density gradient centrifugation.The recipient U251 cells were treated with two kinds of exosomes respectively,and the miR-375 level in recipient U251 cells was detected by q RT-PCR.10.To explore the biological role of miR-375 exosomes with high expression in vivo.U251 cells were used to establish subcutaneous tumor-forming model in nude mice.U251 cells were infected with miR-375 mimic and mimic NC lentivirus to establish stable cell lines.Exosomes derived from two stable cell lines were extracted and purified by ultracentrifugation and density gradient centrifugation.Subcutaneous tumors of nude mice were treated with two kinds of exosomes respectively,and tumor volumes were measured.ResultsExperiment 1: The expression of miR-375 was significantly decreased in the tissues and exosomes of GBM patients1.The expression level of miR-375 was significantly reduced in GBM exosomes.Sequencing results showed that a total of 196 miRNAs were differentially expressed in the6 groups of GBM tissue samples,among which 95 miRNAs were up-regulated and 101 miRNAs were down-regulated.Compared with corresponding paratumoral tissues,the expression of miR-375 in the exosomes of tumor tissues of GBM patients was significantly lower.2.The miR-375 expression were significantly reduced in GBM cell lines and their exosomes.The q RT-PCR results showed that miR-375 levels were significantly lower in four GBM cell lines,A172,U87,U251 and U373,than in the control cell line NHA.miR-375 levels were also significantly lower in the exosomes derived from the four GBM cell lines than in the exosomes derived from the control cell line NHA.Experiment 2: miR-375 negatively regulated the proliferation,migration and invasion of GBM1.Upregulation of miR-375 can significantly inhibit the proliferation ability of GBM cells.The results of CCK-8 cell proliferation assay showed that the absorbance value at450 nm of A172,U87,U251 and U373 cells transfected with miR-375 mimic was significantly lower than that of control group transfected with mimic NC.2.Upregulation of miR-375 can significantly inhibit the migration ability of GBM cells.The cell scratch assay results showed that the cell migration distance of A172,U87,U251 and U373 cells transfected with miR-375 mimic was significantly lower than that of the control group transfected with mimic NC.3.Upregulation of miR-375 can significantly inhibit the invasion ability of GBM cells.The Transwell invasion assay results showed that the number of A172,U87,U251 and U373 cells transfected with miR-375 mimic passing through matrix gel and microporous membrane was significantly lower than the control group transfected with mimic NC.4.Upregulation of miR-375 can significantly inhibit the growth of GBM in vivo.In the model of in situ intracranial tumor formation in nude mice,the tumor volume formed by stable cell lines using miR-375 mimic was significantly smaller than that of the control group using mimic NC.Experiment 3: miR-375 was transferred among tumor cells via exosomes1.The intercellular transmission of miR-375 is active and can be completed without intercellular contact.In co-culture experiment,the q RT-PCR results showed that the miR-375 level in the recipient U251 cells co-cultured with cells stably transfected with a miR-375 mimic was significantly higher than that in the control group co-cultured with cells stably transfected with mimic NC.2.miR-375 is transmitted from cell to cell via exosomes.Non-exosome components do not have this function.q RT-PCR results showed that miR-375 level in recipient U251 cells treated with exosomes of cell lines stably transfected with miR-375 mimics was significantly increased compared with the control group treated with exosomes of cell lines stably transfected with mimic NC.There was no significant difference in miR-375 levels in recipient U251 cells treated with non-exosome components of miR-375 mimic stable cell lines compared with control cells treated with non-exosome components of mimic NC stable cell lines.Experiment 4: Exosome-mediated overexpression of miR-375 significantly inhibited GBM growth in vitro and in vivo1.Exosomes with high expression of miR-375 could significantly inhibit the proliferation of recipient tumor cells.The results of CCK-8 cell proliferation assay showed that the absorbance value of recipient U251 cells treated with exosomes of miR-375 mimic decreased significantly compared with the cells treated with exosomes of mimic NC at 450 nm.2.Exosomes with high expression of miR-375 can significantly inhibit the migration ability of recipient tumor cells.Cell scratch assay results showed that the migration distance of U251 receptor cells treated with exosomes in miR-375 mimic group was significantly shorter than that treated with mimic NC group.3.Exosomes with high expression of miR-375 could significantly inhibit the invasion ability of the recipient tumor cells.Transwell invasion assay results showed that the number of the recipient U251 cells treated with exosomes of miR-375 mimics was significantly lower than that of the cells treated with mimic NC.4.Exosomes with high expression of miR-375 could significantly inhibit tumor growth in vivo.In the nude mouse model of subcutaneous tumorigenesis,tumor volume measurement results showed that the tumor volume of nude mice treated with exosomes of miR-375 mimic group was significantly smaller than that of control group treated with exosomes of mimic NC group.Conclusion(1)The expression level of miR-375 was significantly decreased in GBM and its exosomes.(2)miR-375 affects GBM progression by regulating proliferation,migration and invasion.(3)miR-375 is transmitted via exosomes among tumor cells.(4)Exosome-mediated transfer of highly expressed miR-375 significantly inhibited GBM growth in vitro and in vivo.(5)Exosome miR-375 has great potential as a diagnostic and prognostic marker and a potential treatment for GBM. |
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