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

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100 μmoL/L TMZ）、RES1组（添加50 μmoL/L RES）、RES+TMZ（添加100 μmoL/L TMZ和50 μmoL/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100 μmoL/L TMZ的基础上,转染BRD4过表达质粒（pcDNA BRD4）或加入50 μmoL/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100 μmoL/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。

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

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 100 μmoL/L TMZ), the RES1 group (adding 50 μmoL/L RES) and RES+TMZ group (adding 100 μmoL/L TMZ and 50 μmoL/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 100 μmoL/L TMZ, T98G cells were transfected with BRD4 overexpression plasmid (pcDNA BRD4) or added 50 μmoL/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 100 μmoL/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 resistance (P<0.05). PcDNA BRD4 could reverse the anti-proliferative and pro-apoptotic effects of RES. BRD4 inhibitor JQ1 could inhibit the expression and proliferation of BRD4, Wnt3a/β-catenin and MGMT proteins in T98G cells, and promote apoptosis (P<0.05). LiCl could reverse the anti-proliferative and pro-apoptotic effects of JQ1. RES alone or in combination with JQ1 could activate the inhibitory effect of TMZ on Wnt3a/β-catenin pathway, MGMT expression and cell proliferation in tumors (P<0.05). Conclusion RES can reverse TMZ resistance of glioma T98G cells by downregulating BRD4 and thereby inhibiting the activation of Wnt3a/β-catenin pathway.