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Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma.

ABSTRACT: BACKGROUND:Although temozolomide (TMZ) resistance is a significant clinical problem in glioblastoma (GBM), its underlying molecular mechanisms are poorly understood. In this study, we identified the role of exosomal microRNAs (miRNAs) from TMZ-resistant cells as important mediators of chemoresistance in GBM cells. METHODS:Exosomes were isolated from TMZ-resistant GBM cells and characterized via scanning electron microscopy (SEM). Expression levels of miR-1238 in GBM cell lines and their exosomes, clinical tissues, and sera were evaluated by RT-qPCR. In vitro and in vivo experiments were performed to elucidate the function of exosomal miR-1238 in TMZ resistance in GBM cells. Co-immunoprecipitation assays and western blot analysis were used to investigate the potential mechanisms of miR-1238/CAV1 that contribute to TMZ resistance. FINDINGS:MiR-1238 levels were higher in TMZ-resistant GBM cells and their exosomes than in sensitive cells. Higher levels of miR-1238 were found in the sera of GBM patients than in healthy people. The loss of miR-1238 may sensitize resistant GBM cells by directly targeting the CAV1/EGFR pathway. Furthermore, bioactive miR-1238 may be incorporated into the exosomes shed by TMZ-resistant cells and taken up by TMZ-sensitive cells, thus disseminating TMZ resistance. INTERPRETATION:Our findings establish that miR-1238 plays an important role in mediating the acquired chemoresistance of GBM and that exosomal miR-1238 may confer chemoresistance in the tumour microenvironment. These results suggest that circulating miR-1238 serves as a clinical biomarker and a promising therapeutic target for TMZ resistance in GBM. FUND: This study was supported by the National Natural Science Foundation of China (No·81402056, 81472362, and 81772951) and the National High Technology Research and Development Program of China (863) (No·2012AA02A508).

SUBMITTER: Yin J

PROVIDER: S-EPMC6491393 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma.

Yin Jianxing J Zeng Ailiang A Zhang Zhuoran Z Shi Zhumei Z Yan Wei W You Yongping Y

EBioMedicine 20190324

<h4>Background</h4>Although temozolomide (TMZ) resistance is a significant clinical problem in glioblastoma (GBM), its underlying molecular mechanisms are poorly understood. In this study, we identified the role of exosomal microRNAs (miRNAs) from TMZ-resistant cells as important mediators of chemoresistance in GBM cells.<h4>Methods</h4>Exosomes were isolated from TMZ-resistant GBM cells and characterized via scanning electron microscopy (SEM). Expression levels of miR-1238 in GBM cell lines and ...[more]

PMID: 30917935

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Project description:BackgroundAcquired drug resistance is a constraining factor in clinical treatment of glioblastoma (GBM). However, the mechanisms of chemoresponsive tumors acquire therapeutic resistance remain poorly understood. Here, we aim to investigate whether temozolomide (TMZ) resistance of chemoresponsive GBM was enhanced by long non-coding RNA SBF2 antisense RNA 1 (lncRNA SBF2-AS1) enriched exosomes.MethodLncSBF2-AS1 level in TMZ-resistance or TMZ-sensitive GBM tissues and cells were analyzed by qRT-PCR and FISH assays. A series of in vitro assay and xenograft tumor models were performed to observe the effect of lncSBF2-AS1 on TMZ-resistance in GBM. CHIP assay were used to investigate the correlation of SBF2-AS1 and transcription factor zinc finger E-box binding homeobox 1 (ZEB1). Dual-luciferase reporter, RNA immunoprecipitation (RIP), immunofluorescence and western blotting were performed to verify the relation between lncSBF2-AS1, miR-151a-3p and XRCC4. Comet assay and immunoblotting were performed to expound the effect of lncSBF2-AS1 on DNA double-stand break (DSB) repair. A series of in vitro assay and intracranial xenografts tumor model were used to determined the function of exosomal lncSBF2-AS1.ResultIt was found that SBF2-AS1 was upregulated in TMZ-resistant GBM cells and tissues, and overexpression of SBF2-AS1 led to the promotion of TMZ resistance, whereas its inhibition sensitized resistant GBM cells to TMZ. Transcription factor ZEB1 was found to directly bind to the SBF2-AS1 promoter region to regulate SBF2-AS1 level and affected TMZ resistance in GBM cells. SBF2-AS1 functions as a ceRNA for miR-151a-3p, leading to the disinhibition of its endogenous target, X-ray repair cross complementing 4 (XRCC4), which enhances DSB repair in GBM cells. Exosomes selected from temozolomide-resistant GBM cells had high levels of SBF2-AS1 and spread TMZ resistance to chemoresponsive GBM cells. Clinically, high levels of lncSBF2-AS1 in serum exosomes were associated with poor response to TMZ treatment in GBM patients.ConclusionWe can conclude that GBM cells remodel the tumor microenvironment to promote tumor chemotherapy-resistance by secreting the oncogenic lncSBF2-AS1-enriched exosomes. Thus, exosomal lncSBF2-AS1 in human serum may serve as a possible diagnostic marker for therapy-refractory GBM.

Project description:BackgroundDespite the development of new treatment protocols for glioblastoma (GBM), temozolomide (TMZ) resistance remains a primary hindrance. Previous studies, including our study, have shown that aberrant N6-methyladenosine (m6 A) modification is implicated in GBM pathobiology. However, the roles and precise mechanisms of m6 A modification in the regulation of TMZ resistance in GBM remain unclear.Methodsm6 A individual-nucleotide-resolution cross-linking and immunoprecipitation sequencing (miCLIP-seq) was performed to identify m6 A modification of transcripts in TMZ-resistant and -sensitive tumors. To explore the role of METTL3 in TMZ resistance, TMZ-resistant GBM cells were transfected with METTL3 shRNA or overexpression lentivirus and then assessed by cell viability, tumor sphere formation, and apoptosis assays. An intracranial GBM xenograft model was developed to verify the effect of METTL3 depletion during TMZ treatment in vivo. ATAC-seq, ChIP-qPCR, and dual-luciferase reporter assays were carried out to verify the role of SOX4/EZH2 in the modulation of METTL3 expression upon TMZ treatment.ResultsWe demonstrated that TMZ treatment upregulated the expression of the m6 A methyltransferase METTL3, thereby increasing m6 A modification of histone modification-related gene transcripts. METTL3 is required to maintain the features of GBM stem cells. When combined with TMZ, METTL3 silencing suppressed orthotopic TMZ-resistant xenograft growth in a cooperative manner. Mechanistically, TMZ induced a SOX4-mediated increase in chromatin accessibility at the METTL3 locus by promoting H3K27ac levels and recruiting RNA polymerase II. Moreover, METTL3 depletion affected the deposition of m6 A on histone modification-related gene transcripts, such as EZH2, leading to nonsense-mediated mRNA decay. We revealed an important role of EZH2 in the regulation of METTL3 expression, which was via an H3K27me3 modification-independent manner.ConclusionsOur findings uncover the fundamental mechanisms underlying the interplay of m6 A RNA modification and histone modification in TMZ resistance and emphasize the therapeutic potential of targeting the SOX4/EZH2/METTL3 axis in the treatment of TMZ-resistant GBM.

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Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma.

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Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma.

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