摘要
目的探讨Nei内切核酸酶Ⅷ样蛋白3(NEIL3)在脑胶质瘤中的表达及其对脑胶质瘤细胞生长、迁移、自噬和替莫唑胺耐药性的影响,并揭示其调控机制。方法15例人脑胶质瘤组织样本和癌旁组织样本收集自郑州大学第一附属医院神经外科,通过定量聚合酶链反应(qPCR)实验评估NEIL3在脑胶质瘤组织中表达,通过基因敲减技术降低NEIL3在脑胶质瘤细胞中的表达,提取并纯化NEIL3来源的外泌体,采用细胞计数试剂盒(CCK-8)法和克隆形成实验检测细胞增殖能力,Transwell实验评估细胞迁移能力,免疫印迹和免疫荧光技术检测自噬相关蛋白的表达水平,流式细胞术测定细胞对替莫唑胺的敏感性,此外,通过免疫印迹分析磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路的活性变化,采用t检验比较两组之间的差异,单因素方差分析(ANOVA)用于多组比较。结果脑胶质瘤组织中NEIL3 RNA表达水平(1.00±0.04)明显高于癌旁组织(0.47±0.11),差异有统计学意义(P<0.01)。敲减NEIL3来源的外泌体能够抑制脑胶质瘤细胞的生长[(55.00±0.06)%比(100.00±0.53)%,P<0.01]和集落形成[(49.67±11.55)个比(197.30±44.24)个,P<0.01],抑制脑胶质瘤细胞的自噬,改善脑胶质瘤细胞对替莫唑胺的耐药性,并抑制脑胶质瘤细胞的PI3K/Akt/mTOR信号通路。结论敲低NEIL3来源的外泌体可以抑制脑胶质瘤细胞的迁移和自噬,改善对替莫唑胺的耐药性。
ObjectiveTo investigate the expression of Nei endonucleaseⅧ-like 3(NEIL3)in glioma and its effects on the growth,migration,autophagy,and Temozolomide resistance of glioma cells,as well as to elucidate its regulatory mechanisms.MethodsA total of 15 samples of human glioma tissue and adjacent tissue were collected from the Neurosurgery Department of the First Affiliated Hospital of Zhengzhou University.This study assessed the expression of NEIL3 in glioma tissues via qPCR assays,and utilized gene knockdown techniques to reduce NEIL3 expression in glioma cells and extract and purify NEIL3-derived exosomes.Cell proliferation was assessed using the cell counting kit-8(CCK-8)assay and colony formation assay,and cell migration was evaluated using the Transwell assay.Autophagy-related protein expression levels were detected using immunoblotting and immunofluorescence techniques.The sensitivity of glioma cells to Temozolomide was determined by flow cytometry,and the activity of the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway was analyzed by immunoblotting.T-test was used to compare the differences between two groups,and one-way analysis of variance(ANOVA)was used for multi group comparisons.ResultsThe RNA expression level of NEIL3 in glioma tissues(1.00±0.04)was significantly higher than that in adjacent normal tissues(0.47±0.11,P<0.01).Knockdown of NEIL3-derived exosomes could inhibit the growth[(55.00±0.06)%vs.(100.00±0.53)%,P<0.01]and colony formation(49.67±11.55 vs.197.30±44.24,P<0.01)of glioma cells,suppress autophagy in glioma cells,improve the resistance of glioma cells to temozolomide,and inhibit the PI3K/Akt/mTOR signaling pathway in glioma cells.ConclusionKnockdown of NEIL3-derived exosomes can inhibit glioma cell migration and autophagy and improve Temozolomide sensitivity.
作者
谢祎
闻嘉
Xie Yi;Wen Jia(Department of Neurology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China;Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)
出处
《中华实验外科杂志》
CAS
2024年第8期1755-1758,共4页
Chinese Journal of Experimental Surgery
