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

doi:10.11958/20220566

摘要/Abstract

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。

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

Abstract:

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.

Key words: glioma, drug resistance, neoplasm, Wnt signaling pathway, beta catenin, resveratrol, temozolomide, bromodomain-containing protein 4

中图分类号:

R739.41

图/表 17

图1 TMZ不同敏感细胞株中BRD4、Wnt3a、β-catenin、MGMT蛋白表达免疫印迹图

Fig. 1 Immunoblot of BRD4, Wnt3a, β-catenin and MGMT protein expression in different sensitive cell lines of TMZ

表1 TMZ不同敏感细胞株中BRD4、Wnt3a、β-catenin、MGMT蛋白表达比较（n=6,$\bar{x} \pm s$）

Tab. 1 Comparison of protein expressions of BRD4, Wnt3a, β-catenin and MGMT between different sensitive cell lines of TMZ

细胞系	BRD4	Wnt3a	β-catenin	MGMT
U251	0.38±0.06	0.42±0.07	0.26±0.05	0.63±0.07
U138	0.91±0.11^a	1.23±0.13^a	1.52±0.14^a	1.78±0.12^a
T98G	1.85±0.21^ab	2.09±0.28^ab	2.17±0.26^ab	2.69±0.24^ab
F	166.826^＊＊	125.287^＊＊	189.237^＊＊	249.449^＊＊

图2 不同浓度RES对T98G细胞增殖活性的影响

Fig.2 Effects of different concentrations of RES on the proliferation activity of T98G cells

图3 不同浓度TMZ对T98G细胞增殖活性的影响

Fig.3 The effect of different concentrations of TMZ on the proliferation activity of T98G cells

图4 各组T98G细胞流式凋亡图

Fig.4 Flow cytometry chart of apoptosis of T98G cells in each group

图5 BRD4、Wnt3a、β-catenin、MGMT蛋白表达免疫印迹图

Fig.5 Immunoblot results of BRD4, Wnt3a, β-catenin, MGMT protein expression

表2 RES对T98G细胞增殖活性、凋亡率及BRD4、Wnt3a、β-catenin、MGMT蛋白表达的影响（n=6,$\bar{x} \pm s$）

Tab. 2 Effects of RES on the proliferation activity, apoptosis rate and protein expression of BRD4, Wnt3a,β-catenin and MGMT in T98G cells

组别	增殖活性（%）			凋亡率（%）		BRD4
对照1组	100.00±5.06			9.14±2.13		1.78±0.19
RES1组	51.19±6.71^a			23.19±4.08^a		1.36±0.27^a
RES+TMZ组	25.47±2.63^ab			45.36±6.52^ab		0.74±0.12^b
F	332.660^＊＊			94.239^＊＊		39.929^＊＊
组别		Wnt3a	β-catenin		MGMT
对照1组		1.32±0.18	1.86±0.33		2.19±0.48
RES1组		0.93±0.16^a	1.47±0.12^a		1.68±0.23^a
RES+TMZ组		0.42±0.11^b	0.65±0.18^b		1.04±0.13^b
F		52.305^＊＊	44.096^＊＊		19.909^＊＊

图6 RES对T98G细胞TMZIC50的影响

Fig.6 The effect of RES on TMZIC50 in T98G cells

图7 BRD4、Wnt3a、β-catenin、MGMT蛋白表达免疫印迹图 A：pcDNA NC组;B：pcDNA BRD4组;C：RES2组：D：RES+pcDNA BRD4组。

Fig.7 Western blot assay of BRD4, Wnt3a, β-catenin and MGMT protein expression

表3 各组细胞增殖活性、凋亡率、BRD4、Wnt3a、β-catenin、MGMT蛋白表达比较（n=6,$\bar{x} \pm s$）

Tab. 3 Comparison of cell proliferation activity, apoptosis rate, BRD4, Wnt3a, β-catenin and MGMT protein expression between the four groups

组别	增殖活性（%）	凋亡率（%）	BRD4	Wnt3a	β-catenin	MGMT
pcDNA NC组	100.02±6.18	9.16±1.09	1.78±0.19	1.32±0.18	1.86±0.33	2.07±0.38
pcDNA BRD4组	160.73±15.02^a	2.74±0.16^a	2.53±0.26^a	2.45±0.31^a	2.84±0.37^a	2.95±0.41^a
RES2组	51.23±6.84^a	22.36±5.07^a	1.32±0.28^a	0.92±0.25^a	1.42±0.18^a	1.57±0.15^a
RES+pcDNA BRD4组	93.42±7.15^bc	13.75±1.26^bc	1.96±0.41	1.76±0.19^bc	2.15±0.37^bc	2.51±0.17^bc
F	134.977^＊＊	57.308^＊＊	17.162^＊＊	45.382^＊＊	20.542^＊＊	23.081^＊＊

图8 T98G细胞流式凋亡图

Fig.8 Flow cytometry chart of apoptosis of T98G cells

图9 T98G细胞流式凋亡图

Fig.9 Flow cytometry chart of apoptosis of T98G cells

图10 BRD4、Wnt3a、β-catenin、MGMT蛋白表达免疫印迹图

Fig.10 Immunoblot results of BRD4, Wnt3a, β-catenin and MGMT protein expression

表4 各组增殖活性、凋亡率、BRD4、Wnt3a、β-catenin、MGMT蛋白表达的比较（n=6,$\bar{x} \pm s$）

Tab. 4 Comparison of proliferation activity, apoptosis rate, BRD4, Wnt3a, β-catenin and MGMT protein expression between the three groups

组别	增殖活性（%）	凋亡率（%）	BRD4	Wnt3a	β-catenin	MGMT
对照2组	100.00±6.43	11.43±3.15	1.69±0.13	1.31±0.19	1.84±0.32	2.15±0.23
JQ1组	51.19±7.42^a	29.15±3.87^a	1.28±0.17^a	0.89±0.07^a	1.36±0.17^a	1.82±0.09^a
JQ1+LiCl组	98.23±7.51^b	13.46±3.21^b	1.25±0.19	1.25±0.09^b	1.79±0.12^b	2.09±0.11^b
F	92.280^＊＊	48.089^＊＊	13.282^＊＊	18.916^＊＊	8.603^＊＊	7.609^＊＊

图11 瘤体内Ki67免疫组化染色图（标尺：50 μm）

Fig.11 Immunohistochemical staining showing Ki67 in tumor

图12 瘤体中BRD4、Wnt3a、β-catenin、MGMT蛋白表达免疫印迹图 A：对照治疗组;B：TMZ治疗组;C：RES+TMZ治疗组;D：RES+TMZ+JQ1治疗组。

Fig.12 Immunoblot results of BRD4, Wnt3a, β-catenin and MGMT protein expression in tumor

表5 瘤体中Ki67、BRD4、Wnt3a、β-catenin、MGMT蛋白表达比较（n=5,$\bar{x} \pm s$）

Tab. 5 Comparison of Ki67, BRD4, Wnt3a, β-catenin and MGMT protein expressions in tumors between the four groups

组别	Ki67（光密度/mm²）	BRD4	Wnt3a	β-catenin	MGMT
对照治疗组	2.13±0.18	1.57±0.10	1.83±0.14	1.25±0.16	2.13±0.17
TMZ治疗组	2.10±0.21	1.53±0.12	1.82±0.18	1.23±0.15	2.10±0.14
RES+TMZ治疗组	1.47±0.13^a	1.18±0.15^a	1.27±0.12^a	0.96±0.12^a	1.71±0.19^a
RES+TMZ+JQ1治疗组	0.85±0.09^ab	0.61±0.11^ab	0.53±0.06^ab	0.48±0.07^ab	1.06±0.12^ab
F	72.557^＊＊	66.924^＊＊	107.569^＊＊	38.160^＊＊	50.141^＊＊

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