白藜芦醇通过BRD4调控Wnt/β-catenin通路逆转胶质瘤细胞替莫唑胺耐药的机制研究  

Study on the mechanism of resveratrol reversing temozolomide resistance in glioma cells by regulating Wnt/β-catenin pathway through BRD4

作　　者：李振江 孙德超 孔晨旭 耿亚东 徐晨阳 丁炳谦 LI Zhenjiang;SUN Dechao;KONG Chenxu;GENG Yadong;XU Chenyang;DING Bingqian(Department of Neurosurgery,Huaihe Hospital of Henan University,Kaifeng 475000,China)

机构地区：[1]河南大学淮河医院神经外科,475000

出　　处：《天津医药》2022年第10期1043-1050,共8页Tianjin Medical Journal

基　　金：河南省科技攻关项目(222102310522)。

摘　　要：目的探究白藜芦醇(RES)逆转胶质瘤细胞替莫唑胺(TMZ)耐药的作用是否与通过溴结合域蛋白4(BRD4)调控Wnt/β-链蛋白(β-catenin)通路有关。方法取人神经胶质瘤TMZ低敏细胞株(U138)、高敏细胞株(U251)、耐药株(T98G),Western blot法检测3种细胞株中BRD4、Wnt3a、β-catenin、TMZ耐药蛋白(MGMT)蛋白表达。取T98G细胞株分为对照1组(添加100μmoL/L TMZ)、RES1组(添加50μmoL/L RES)、RES+TMZ(添加100μmoL/L TMZ和50μmoL/L RES)组,用CCK-8法、流式细胞术检测各组细胞增殖、凋亡情况;Western blot法检测各组BRD4、Wnt3a、β-catenin、MGMT蛋白表达。为分析BRD4过表达对TMZ耐药性的影响,在添加100μmoL/L TMZ的基础上,转染BRD4过表达质粒(pcDNA BRD4)或加入50μmoL/L RES分别作为pcDNA NC组、pcDNA BRD4组、RES2组、RES+pcDNA BRD4组。为验证BRD4对Wnt3a/β-catenin通路的调控作用,在添加100μmoL/L TMZ的基础上,加入BRD4抑制剂JQ1和Wnt3a/β-catenin通路激活剂LiCl,分为对照2组、JQ1组、JQ1+LiCl组。将T98G细胞接种于裸鼠左肩胛区,给予RES、TMZ和(或)JQ1治疗,检测瘤体中Ki67,BRD4、MGMT及Wnt3a/β-catenin蛋白表达。结果与U251细胞相比,U138、T98G中BRD4、Wnt3a、β-catenin及MGMT表达均依次升高(P<0.05)。与对照1组相比,RES干预可抑制T98G细胞BRD4、Wnt3a/β-catenin、MGMT蛋白表达及增殖,促进凋亡,逆转细胞的耐药性(P<0.05)。pcDNA BRD4可逆转RES的抗增殖、促凋亡等上述作用。BRD4抑制剂JQ1可抑制T98G细胞BRD4、Wnt3a/β-catenin、MGMT蛋白表达及增殖,促进凋亡(P<0.05);LiCl可逆转JQ1的抗增殖、促凋亡作用。RES单独或与JQ1联合治疗,可激活TMZ对瘤体内Wnt3a/β-catenin通路、MGMT表达和细胞增殖的抑制作用(P<0.05)。结论RES可能通过下调BRD4,进而抑制Wnt3a/β-catenin通路活化,实现对胶质瘤T98G细胞TMZ耐药性的逆转。Objective To explore whether the effect of resveratrol in reversing temozolomide(TMZ)resistance in glioma cells was related to the regulation of bromodomain-containing protein 4(BRD4)and Wnt/β-catenin pathways.Methods Human glioma TMZ low-sensitivity cell line(U138),high-sensitivity cell line(U251)and drug-resistant cell line(T98G)were obtained,and Western blot assay was performed to detect the protein expressions of BRD4,Wnt3a,β-catenin and TMZ drug resistance protein(MGMT).T98G cell line was taken and divided into the control group 1(adding 100μmoL/L TMZ),the RES1 group(adding 50μmoL/L RES)and RES+TMZ group(adding 100μmoL/L TMZ and 50μmoL/L RES).Cell proliferation and apoptosis were measured by CCK-8 method and flow cytometry.Protein expressions of BRD4,Wnt3a,β-catenin and MGMT were measured by Western blot assay.In order to analyze the effect of BRD4 overexpression on TMZ resistance,on the basis of adding 100μmoL/L TMZ,T98G cells were transfected with BRD4 overexpression plasmid(pcDNA BRD4)or added 50μmoL/L RES,and cells were divided into the pcDNA NC group,the pcDNA BRD4 group,the RES2 group and the RES+pcDNA BRD4 group.In order to verify the regulatory effect of BRD4 on Wnt3a/β-catenin pathway,on the basis of adding 100μmoL/L TMZ,BRD4 inhibitor JQ1 and the Wnt3a/β-catenin pathway activator LiCl were added,and cells were divided into the control 2 groups,the JQ1 group and the JQ1+LiCl group.T98G cells were inoculated into the left scapular region of nude mice,and treated with RES,TMZ and/or JQ1.Protein expressions of Ki67,BRD4, MGMT and Wnt3a/β -catenin in tumor were measured. Results Compared with the high-susceptibility strain (U251), the expressions of BRD4, Wnt3a, β-catenin and MGMT increased in sequence in the low-susceptibility strain (U138) and the drug-resistant strain (T98G) (P<0.05). Compared with the control group 1, RES intervention could inhibit the expression and proliferation of BRD4, Wnt3a/β-catenin and MGMT proteins in T98G cells, promote apoptosis and reverse cell drug resista

关 键 词：神经胶质瘤 抗药性 肿瘤 Wnt信号通路 Β连环素 白藜芦醇 替莫唑胺 溴结合域蛋白4 

分 类 号：R739.41[医药卫生—肿瘤]