Extracellular vesicles derived from hypoxic glioma stem-like cells confer temozolomide resistance on glioblastoma by delivering miR-30b-3p.
Extracellular vesicles derived from hypoxic glioma stem-like cells confer temozolomide resistance on glioblastoma by delivering miR-30b-3p.
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源自缺氧胶质瘤干细胞的细胞外囊泡通过传递 miR-30b-3p 赋予胶质母细胞瘤替莫唑胺耐药性
作者：
Yin J;Ge X;Shi Z;Yu C;Lu C;Wei Y;Zeng A;Wang X;Yan W;Zhang J;You Y
原理：胶质瘤干细胞样细胞（GSCs）有助于胶质瘤对替莫唑胺（TMZ）的耐药性，尽管其机制尚未明确。方法：采用体外功能实验（集落形成实验、流式细胞术分析、TUNEL实验）评估缺氧GSCs的细胞外囊泡（ev）促进胶质母细胞瘤（GBM）细胞对TMZ耐药的能力。采用RNA测序和定量逆转录pcr技术鉴定缺氧ev的功能miRNA。采用染色质免疫沉淀法分析HIF1α和STAT3对mirna的转录调控。采用RIP和RNA下拉实验验证了hnrnpa2b1介导的miRNA包装到ev中的效果。通过体内实验和临床样本分析，验证了缺氧GSCs中EV miR-30b-3p的功能。结果：低氧gsc衍生的ev比常氧gsc衍生的ev对GBM化学耐药的影响更大。miRNA分析显示，miR-30b-3p在缺氧GSCs的ev中显著上调。此外，HIF1α和STAT3转录诱导miR-30b-3p表达。RNA免疫沉淀和RNA下拉实验显示，miR-30b-3p与hnRNPA2B1的结合促进了其转移到ev中。ev包装的miR-30b-3p （EV-miR-30b-3p）直接靶向RHOB，导致体外和体内细胞凋亡减少，增殖增加。我们的研究结果提供了证据，证明脑脊液中的miR-30b-3p可能是预测TMZ耐药性的潜在生物标志物。结论：我们的研究结果表明，靶向EV-miR-30b-3p可能为GBM提供潜在的治疗策略。
Rationale: Glioma stem-like cells (GSCs) contribute to temozolomide (TMZ) resistance in gliomas, although the mechanisms have not been delineated. Methods: In vitro functional experiments (colony formation assay, flow cytometric analysis, TUNEL assay) were used to assess the ability of extracellular vesicles (EVs) from hypoxic GSCs to promote TMZ resistance in glioblastoma (GBM) cells. RNA sequencing and quantitative Reverse Transcription-PCR were employed to identify the functional miRNA in hypoxic EVs. Chromatin immunoprecipitation assays were performed to analyze the transcriptional regulation of miRNAs by HIF1α and STAT3. RIP and RNA pull-down assays were used to validate the hnRNPA2B1-mediated packaging of miRNA into EVs. The function of EV miR-30b-3p from hypoxic GSCs was verified by in vivo experiments and analysis of clinical samples. Results: Hypoxic GSC-derived EVs exerted a greater effect on GBM chemoresistance than those from normoxic GSCs. The miRNA profiling revealed that miR-30b-3p was significantly upregulated in the EVs from hypoxic GSCs. Further, HIF1α and STAT3 transcriptionally induced miR-30b-3p expression. RNA immunoprecipitation and RNA-pull down assays revealed that binding of miR-30b-3p with hnRNPA2B1 facilitated its transfer into EVs. EV-packaged miR-30b-3p (EV-miR-30b-3p) directly targeted RHOB, resulting in decreased apoptosis and increased proliferation in vitro and in vivo. Our results provided evidence that miR-30b-3p in CSF could be a potential biomarker predicting resistance to TMZ. Conclusion: Our findings indicated that targeting EV-miR-30b-3p could provide a potential treatment strategy for GBM.