细胞外囊泡携带 miR-27a-3p 通过靶向 BTG2 促进胶质母细胞瘤对替莫唑胺的耐药性。
Extracellular vesicles carry miR-27a-3p to promote drug resistance of glioblastoma to temozolomide by targeting BTG2.
机构信息
Department of Neurosurgery, The First Affiliated Hospital of Xi'an Jiaotong University, No. 277 Yanta West Road, Xi'an, 710061, Shaanxi, People's Republic of China.
Department of Neurosurgery, Xi'an International Medical Center Hospital, Xi'an, 710100, Shaanxi, People's Republic of China.
出版信息
Cancer Chemother Pharmacol. 2022 Feb;89(2):217-229. doi: 10.1007/s00280-021-04392-1. Epub 2022 Jan 17.
OBJECTIVE
Glioblastoma (GBM) is the most common central nervous system tumor. Temozolomide (TMZ) is a commonly used drug for GBM management. This study explored the mechanism of extracellular vesicles (EVs) regulating TMZ-resistance in GBM.
METHODS
LN229 cells were inducted into TMZ-resistant LN229r strain by stepwise induction. After the intervention of miR-27a-3p expression, cell viability of GBM cells treated with different concentrations of TMZ was detected by MTT and IC50 value was calculated. Cell proliferation and apoptosis were detected by colony formation and flow cytometry. EVs extracted from LN18 cells were identified and the internalization of EVs by LN229r cells was evaluated. The 100 μmol/L TMZ-treated LN229r cells were treated with EVs or EVs with downregulated miR-27a-3p to verify the effect of EVs-carried miR-27a-3p on TMZ resistance. The binding relation between BTG2 and miR-27a-3p was verified. miR-27a-3p and BTG2 expressions in GBM cells and EVs were detected by RT-qPCR. The BTG2 effect on TMZ-resistance in GBM was verified. The xenograft tumor nude mouse model was established by injecting LN229r cells and treated with EVs and 100 μmol/L TMZ.
RESULTS
miR-27a-3p was highly expressed in LN229r cells. IC50 value and proliferation of LN229r cells with silenced miR-27a-3p were decreased and apoptosis was increased, indicating that miR-27a-3p silencing reduced the drug-resistant cell LN229r resistance to TMZ. LN18-derived EVs could be internalized by LN229r cells, and release its encapsulated miR-27a-3p into LN229r cells and increase miR-27a-3p expression. EV treatment increased LN229r cell proliferation and reduced apoptosis, while EVs with silenced miR-27a-3p showed the opposite trend. miR-27a-3p targeted BTG2. BTG2 overexpression reduced LN229r cell resistance to TMZ. In vivo, after EVs treatment, tumor volume and weight, Ki67-positive rate, and miR-27a-3p were increased, while BTG2 expression was decreased.
CONCLUSION
GBM-derived EVs were internalized by GBM cells, released miR-27a-3p into GBM cells, upregulated miR-27a-3p expression, and targeted BTG2, thus promoting TMZ resistance.
目的
胶质母细胞瘤（GBM）是最常见的中枢神经系统肿瘤。替莫唑胺（TMZ）是一种常用于 GBM 治疗的药物。本研究探讨了细胞外囊泡（EVs）调节 GBM 中 TMZ 耐药的机制。
方法
通过逐步诱导，将 LN229 细胞诱导为 TMZ 耐药的 LN229r 株。在 miR-27a-3p 表达干预后，通过 MTT 检测不同浓度 TMZ 处理的 GBM 细胞的细胞活力，并计算 IC50 值。通过集落形成和流式细胞术检测细胞增殖和凋亡。鉴定 LN18 细胞中提取的 EVs，并评估 LN229r 细胞对 EVs 的内化。用 100μmol/L TMZ 处理 LN229r 细胞，然后用 EVs 或下调 miR-27a-3p 的 EVs 处理，以验证 EV 携带的 miR-27a-3p 对 TMZ 耐药性的影响。验证 BTG2 与 miR-27a-3p 的结合关系。通过 RT-qPCR 检测 GBM 细胞和 EVs 中 miR-27a-3p 和 BTG2 的表达。验证 BTG2 对 GBM 中 TMZ 耐药性的影响。通过注射 LN229r 细胞并分别用 EVs 和 100μmol/L TMZ 处理，建立裸鼠异种移植肿瘤模型。
结果
miR-27a-3p 在 LN229r 细胞中高表达。沉默 miR-27a-3p 的 LN229r 细胞的 IC50 值和增殖降低，凋亡增加，表明 miR-27a-3p 沉默降低了耐药细胞 LN229r 对 TMZ 的耐药性。LN18 衍生的 EVs 可被 LN229r 细胞内化，并将其包裹的 miR-27a-3p 释放到 LN229r 细胞中，从而增加 miR-27a-3p 的表达。EV 处理增加了 LN229r 细胞的增殖，减少了凋亡，而沉默 miR-27a-3p 的 EVs 则表现出相反的趋势。miR-27a-3p 靶向 BTG2。BTG2 过表达降低了 LN229r 细胞对 TMZ 的耐药性。在体内，经 EVs 处理后，肿瘤体积和重量、Ki67 阳性率和 miR-27a-3p 增加，而 BTG2 表达减少。
结论
GBM 来源的 EVs 被 GBM 细胞内化，将 miR-27a-3p 释放到 GBM 细胞中，上调 miR-27a-3p 的表达，并靶向 BTG2，从而促进 TMZ 耐药性。
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