二甲双胍增加U87 MG人胶质母细胞瘤细胞外泌体的生物发生和分泌：治疗耐药性的可能机制。,Archives of Medical Research

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二甲双胍增加U87 MG人胶质母细胞瘤细胞外泌体的生物发生和分泌：治疗耐药性的可能机制。
Archives of Medical Research ( IF 4.7 ) Pub Date : 2020-10-13 , DOI: 10.1016/j.arcmed.2020.10.007
Hamid Soraya , Neda Abbaspour Sani , Nassrollah Jabbari , Jafar Rezaie

背景

多形胶质母细胞瘤（GBM）是最常见和侵略性的脑肿瘤。二甲双胍是一种抗糖尿病药，可以抑制肿瘤细胞。GBM细胞的外泌体有助于细胞间通讯，肿瘤侵袭性和治疗抗性。我们研究了二甲双胍对U87 MG细胞外泌体分泌途径的影响。

方法

使用MTT分析研究了针对二甲双胍的细胞存活率。通过实时PCR计算miRNA-21，miRNA-155和miRNA-182的表达，以及与外泌体生物发生和分泌有关的基因，如Rab27a，Rab27b，Rab11，CD63和Alix。通过蛋白质印迹分析CD63蛋白的表达，而通过流式细胞术监测CD63蛋白的亚细胞分布。外来体的特征在于透射和扫描电子显微镜以及流式细胞仪。使用乙酰胆碱酯酶活性测定法和ELISA测定外泌体的量。使用ELISA评估自噬标记物LC3和P62的表达。

结果

数据显示二甲双胍可降低细胞存活率以及miRNA-21，miRNA-155和miRNA-182的表达（p <0.05）。在处理过的细胞中，Rab27a，Rab27b，Rab11，CD63和Alix的表达以及CD63的蛋白水平上调（p <0.05）。同时，流式细胞仪分析显示，经处理的细胞表面CD63 /总CD63比例增加（p <0.05）。我们发现处理过的细胞的外泌体的乙酰胆碱酯酶活性和CD63蛋白增加（p <0.05）。LC3和P62的表达不受二甲双胍的影响（p > 0.05）。

结论

数据表明二甲双胍可以促进U87 MG细胞的外泌体生物发生和分泌，从而提出针对二甲双胍的治疗反应。

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Metformin Increases Exosome Biogenesis and Secretion in U87 MG Human Glioblastoma Cells: A Possible Mechanism of Therapeutic Resistance

Background

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Metformin, an anti-diabetic drug, can suppress tumor cells. Exosomes from GBM cells contribute to intercellular communication, tumor aggressiveness, and therapeutic resistance. We studied the effect of metformin on the exosomal secretory pathway in U87 MG cells.

Methods

Cell survival against metformin was investigated using MTT assay. Expression of miRNA-21, miRNA-155, and miRNA-182, as well as the genes involved in exosome biogenesis and secretion such as Rab27a, Rab27b, Rab11, CD63, and Alix were calculated by real time-PCR. The expression of CD63 protein was analyzed by western blotting, while the subcellular distribution of CD63 protein was monitored by flow cytometry. Exosomes were characterized by transmission and scanning electron microscopes, and flow cytometry. Amount of exosomes was assayed using acetylcholinesterase activity assay and ELISA. The expression of autophagic markers LC3 and P62 were assessed using ELISA.

Results

Data showed that metformin decreased cell survival and expression of miRNA-21, miRNA-155, and miRNA-182 (p <0.05). Expression of Rab27a, Rab27b, Rab11, CD63, and Alix as well as protein level of CD63 up-regulated in treated cells (p <0.05). Concurrently, flow cytometry analysis showed that surface CD63/total CD63 ratio was increased in treated cells (p <0.05). We found that acetylcholinesterase activity and CD63 protein of exosomes from treated cells increased (p <0.05). The expression of LC3 and P62 was not affected by metformin (p >0.05).