CircGFRA1调节miR-138-5p/ANXA2轴对脑胶质瘤细胞替莫唑胺耐药性的影响  

Impact of CircGFRA1 on temozolomide resistance in glioma cells by regulating miR-138-5p/ANXA2 axis

作　　者：王启军 汪惊涛 代兴亮 WANG Qi-Jun;WANG Jing-Tao;DAI Xing-Liang(Department of Neurosurgery,Liu'an Hospital Affiliated to Anhui Medical University,Liu'an 237005,Anhui,China)

机构地区：[1]安徽医科大学附属六安医院神经外科,安徽六安237005 [2]安徽医科大学第一附属医院神经外科,安徽六安237005

出　　处：《中国老年学杂志》2024年第22期5491-5497,共7页Chinese Journal of Gerontology

基　　金：国家自然科学基金项目(81702457)。

摘　　要：目的探究环状RNA胶质细胞系源性神经营养因子受体(CircGFRA)1调节miR-138-5p/ANXA2轴对脑胶质瘤细胞替莫唑胺(TMZ)耐药性的影响。方法实时荧光定量聚合酶链反应(qRT-PCR)和Western印迹分别检测脑胶质瘤细胞系U251及U251/TMZ细胞中CircGFRA1、miR-138-5p表达及ANXA2蛋白表达;将10μg/ml TMZ处理的U251/TMZ细胞分为TMZ+U251/TMZ组、TMZ+U251/TMZ+si-NC组、TMZ+U251/TMZ+si-CircGFRA1组、TMZ+U251/TMZ+si-CircGFRA1+anti-NC组、TMZ+U251/TMZ+si-CircGFRA1+anti-miR-138-5p组,qRT-PCR检测细胞中CircGFRA1、miR-138-5p表达;MTT法检测U251/TMZ细胞生长抑制率;Transwell法检测U251/TMZ细胞迁移、侵袭情况;流式细胞术检测U251/TMZ细胞凋亡情况;Western印迹检测ANXA2及凋亡相关蛋白表达;双荧光素酶报告实验检测CircGFRA1、miR-138-5p、ANXA2靶向关系。结果U251/TMZ组较U251组CircGFRA1、ANXA2显著上调,miR-138-5p显著下调(P<0.05),且在5~80μg/ml TMZ作用下,U251/TMZ组生长抑制率均显著下降,IC50显著升高(P<0.05),筛选出较适浓度10μg/ml用于后续实验。与TMZ+U251/TMZ组、TMZ+U251/TMZ+si-NC组相比,TMZ+U251/TMZ+si-CircGFRA1组CircGFRA1及ANXA2、Bcl-2水平、迁移和侵袭细胞数量显著降低,miR-138-5p水平、细胞生长抑制率、凋亡率、Bax、cleaved-Caspase-3水平显著升高(P<0.05);与TMZ+U251/TMZ+si-CircG-FRA1组、TMZ+U251/TMZ+si-CircGFRA1+anti-NC组相比,TMZ+U251/TMZ+si-CircGFRA1+anti-miR-138-5p组显著升高了ANXA2水平及迁移、侵袭能力,显著降低了miR-138-5p水平及细胞生长抑制率、凋亡率(P<0.05);CircGFRA1靶向调控miR-138-5p/ANXA2轴。结论沉默CircGFRA1可能通过上调miR-138-5p来抑制ANXA2表达进而调控TMZ耐药脑胶质瘤细胞的TMZ耐药性。Objective To explore the impact of circular RNA recombinant glial cell line derived neurotrophic factor receptor alpha(CircGFRA)1 on temozolomide(TMZ)resistance in glioma cells by regulating miR-138-5p/ANXA2 axis.Methods Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expressions of CircGFRA1,miR-138-5p and ANXA2 protein in glioma cell lines U251 and U251/TMZ cells,respectively;U251/TMZ cells treated with 10μg/ml TMZ were divided into TMZ+U251/TMZ group,TMZ+U251/TMZ+si-NC group,TMZ+U251/TMZ+si-CircGFRA1 group,TMZ+U251/TMZ+si-CircGFRA1+anti-NC group,and TMZ+U251/TMZ+si-CircGFRA1+anti-miR-138-5p group,qRT-PCR was applied to detect the expressions of CircGFRA1 and miR-138-5p in cells;MTT assay was dopted to detect the growth inhibition rate of U251/TMZ cells;Transwell method was used to detect the migration and invasion of U251/TMZ cells;flow cytometry was applied to detect apoptosis of U251/TMZ cells;Western blot was used to detect the expression of ANXA2 and apoptosis-related proteins;dual-luciferase reporter assay was applied to detect the targeting relationship of CircGFRA1,miR-138-5p,and ANXA2.Results Compared with U251 group,CircGFRA1 and ANXA2 were obviously up-regulated in U251/TMZ group,and miR-138-5p was obviously down-regulated(P<0.05),and under the action of 5~80μg/ml TMZ,the growth inhibition rate of U251/TMZ group was obviously decreased,IC50 was significantly increased(P<0.05),the optimal concentration of 10μg/ml was screened out for subsequent experiments.Compared with TMZ+U251/TMZ group and TMZ+U251/TMZ+si-NC group,the levels of CircGFRA1,ANXA2 and Bcl-2,and the numbers of migrating and invasive cells were obviously decreased,the level of miR-138-5p,cell growth inhibition rate,apoptosis rate,and the levels of Bax,cleaved-Caspase-3 were obviously increased in TMZ+U251/TMZ+si-CircGFRA1 group(P<0.05);compared with TMZ+U251/TMZ+si-CircGFRA1 group and TMZ+U251/TMZ+si-CircGFRA1+anti-NC group,the level of ANXA2,abilities of migration and invasion of cell

关 键 词：环状RNA胶质细胞系源性神经营养因子受体(CircGFRA)1 miR-138-5p ANXA2 替莫唑胺 脑胶质瘤 耐药性 

分 类 号：R739.41[医药卫生—肿瘤]