Exosomal Transfer Of MiR-1238 Contributes To Temozolomide-resistance In Glioblastoma

Research background:Glioma is the most common primary intracranial tumor in adults.Glioblastoma accounts for more than half of the total subtype of glioma.Because of the aggressive and heterogeneous characteristics of GBM,the prognosis of patients is extremely poor.Surgical resection combined with postoperative radiotherapy and chemotherapy is the standard treatment for malignant glioma.Temozolomide is a second-generation oral alkylating agent,which is used as a first-line chemotherapy for glioma due to its lethal effect on malignant glioma cells and its easy passage through the blood-brain barrier.However,temozolomide resistance has become a difficult and urgent clinical problem,and its potential molecular mechanism remains to be further understood.Methods:1.TMZ resistant cell lines were constructed by stepwise induction method,and CCK8 and colony formation assays were used to verify the drug resistance of the cells.2.qRT-PCR were used to seesee the expression levels of miR-1238 in GBM cells,GBM specimens and serum.3.Exosomes were isolated from TMZ resistant GBM cells and characterized via scanning electron microscope(SEM),NanoSight particle-tracking assay(NTA) and western blot.4.CCK-8,colony formation assays and flow cytometric analysis were used to assess the torelance of GBM cells to TMZ after treatment with resistant exosomes.5.Prediction software,western blot and double luciferase reporter gene analysis were employed to identify potential target genes of mir-1238(CAV1).6.CCK-8,colony formation assays and flow cytometric analysis were used to assess the influence of miR-1238/CAV1 axis to GBM cells.7.In vivo experiments were performed to assess the functional effect of exosomal miR-1238 onTMZ resistance.Results:1.We successfully constructed wo TMZ resistant cell lines,U251 r and N3 r.U251r and N3 r exhibit about a 6-or 8-fold change of resistance to TMZ compared with U251s and N3 s,respectively.MiR-1238 was highly expressed in both recurrent GBM samples and TMZ resistant cell lines.2.The expression level of extracellular miR-1238 was positively correlated with TMZ resistance.The expression levels of miR-1238 in the serum of patients with recurrent GBM were higher than that of patients with primary GBM.3.The TMZ resistant cell-derived exosomes could encapsulate miR-1238 to sensitive cells.4.Exosomes from TMZ resistant cells could significantly enhance the drug resistance of sensitive cells and miR-1238 plays a key role in this process.5.CAV1 is a potential target gene of miR-1238.6.miR-1238 /CAV1 axis could regulate TMZ resistance of GBM cells.7.Exosomes transfer of miR-1238 from TMZ resistant cells could enhance GBM resistance to TMZ in vivo.Conclusions:1.The levels of miR-1238 in serum could predicte the reaction of GBM patients to TMZ treatment.2.Exosomal miR-1238 from TMZ resistant cells could enhance the chemoresistance of recipient cells.3.MiR-1238 regulates TMZ resistance of GBM cells by directly targeting CAV1.