Circ_EPHB4通过miR-424-5p/Wnt3信号轴调控胶质瘤细胞对替莫唑胺的化疗敏感性

doi:10.12122/j.issn.1673-4254.2025.05.06

摘要/Abstract

摘要：

目的探讨Circ_EPHB4通过miR-424-5p/Wnt3信号轴调控胶质瘤细胞替莫唑胺敏感性的机制。方法收集我院2019年1月~2021年12月25例原发性胶质瘤患者和25例经替莫唑胺（TMZ）基化疗治愈后的复发性胶质瘤患者组织标本。qRT-PCR检测Circ_EPHB4表达敲低及敲低对照组细胞中circ_EPHB4、miR-424-5p及Wnt3 mRNA的表达水平。Western blotting检测Wnt3蛋白表达水平。细胞活力、克隆形成和细胞凋亡分别通过CCK-8、克隆形成和流式细胞仪检测评估。采用双荧光素酶报告和RNA免疫沉淀（RIP）试验验证circ_EPHB4、miR-424-5p和Wnt3间的靶向调控关系。皮下成瘤实验评估circ_EPHB4对胶质瘤肿瘤形成能力的影响。结果胶质瘤组织和细胞中circ EPHB4的表达明显高于正常神经组织和星形胶质细胞（P=0.014）。与si-NC对照组相比，si-Circ_EPHB4降低了TMZ耐药胶质瘤细胞中circ_EPHB4表达水平（A172：P=0.008；SHG44：P=0.009）。circ_EPHB4表达敲低降低了TMZ耐药胶质瘤细胞对TMZ的IC₅₀值（A172：P=0.012；SHG44：P=0.022），抑制了胶质瘤细胞克隆形成（A172：P=0.004；SHG44：P=0.006），并促进胶质瘤细胞凋亡（A172：P=0.002；SHG44：P=0.00）。胶质瘤组织中miR-424-5p和circ_EPHB4表达呈负相关（r=-0.556，P=0.011）。miR-424-5p表达敲低，逆转了circ_EPHB4表达敲低引起的IC₅₀值下降（P=0.001）、克隆形成抑制（P=0.016）和细胞凋亡促进作用（P=0.001）。此外，miR-424-5p表达敲低，还削弱了circ_EPHB4表达敲低对PCNA、P-gp、MRP1和bax表达的影响（P=0.004）。结论 circ_EPHB4通过“海绵吸附”miR-424-5p调控Wnt3表达，由此调节TMZ耐药胶质瘤细胞克隆形成、细胞凋亡和TMZ敏感性，circ_EPHB4是逆转胶质瘤耐药的潜在靶点。

关键词: 胶质瘤, 化疗抵抗, circ_EPHB4, miR-424-5p, Wnt3

Abstract:

Objective To investigate the mechanism by which circ_EPHB4 regulates temozolomide (TMZ) sensitivity of glioma cells through the miR-424-5p/Wnt3 signal axis. Methods We detected the expression levels of circ_EPHB4, miR-424-5p and Wnt3 mRNA in glioma specimens from 25 patients with primary glioma and 25 patients experiencing relapse following temozolomide-based chemotherapy and in TMZ-sensitive and -resistant glioma A172 and SHG44 cells with circ_EPHB4 knockdown using qRT-PCR, and Wnt3 protein expression level was detected with Western blotting. Cell viability, colony-forming ability, and apoptosis of the cells with circ_EPHB4 knockdown were assessed, and the targeted regulation relationship between circ_EPHB4, miR-424-5p, and Wnt3 was verified by dual luciferase reporter assay and RNA immunoprecipitation (RIP) experiments. The effect of circ_EPHB4 knockdown on tumorigenesis of glioma cells was evaluated in subcutaneous tumor-bearing nude mouse models. Results The expression of circ_EPHB4 was significantly increased in glioma tissues and cells as compared with normal neural tissues and astrocytes (P=0.014). In TMZ-resistant glioma cells, circ_EPHB4 knockdown resulted in an obvious reduction of IC50 value of TMZ, inhibited cell colony formation, and promoted cell apoptosis, and these effects were reversed by miR-424-5p knockdown. The expressions of miR-424-5p and circ_EPHB4 were negatively correlated in glioma tissues (P=0.011). MiR-424-5p knockdown also attenuated the effect of circ_EPHB4 knockdown on expressions of PCNA, P-gp, MRP1 and bax. Conclusion Circ_EPHB4 regulates Wnt3 expression through "sponge adsorption" of miR-424-5p, thereby modulating TMZ-resistant glioblastoma cell clonogenesis, apoptosis, and TMZ sensitivity, suggesting the potential of circ_EPHB4 as a therapeutic target for reversing drug resistance of gliomas.

Key words: glioma, chemoresistance, circ_EPHB4, miR-424-5p, Wnt3

廖宇翔, 刘景平, 刘博, 费喜云, 金晨. Circ_EPHB4通过miR-424-5p/Wnt3信号轴调控胶质瘤细胞对替莫唑胺的化疗敏感性[J]. 南方医科大学学报, 2025, 45(5): 942-953.

Yuxiang LIAO, Jingping LIU, Bo LIU, Xiyun FEI, Chen JIN. Circ_EPHB4 regulates temozolomide sensitivity in glioma cells through the miR-424-5p/Wnt3 axis[J]. Journal of Southern Medical University, 2025, 45(5): 942-953.

图/表 9

图1 Circ_EPHB4的表达与胶质瘤TMZ敏感性的关系

Fig.1 Relationship between circ_EPHB4 expression and TMZ sensitivity of glioma cells. A: Comparison of circ_EPHB4 expression in primary glioma tissues (sensitive), recurrent glioma tissues (resistant), and healthy brain tissues (normal). B: Comparison of c irc_EPHB4 expression in HBE, A172, A172/TMZ, SHG44, and SHG44/TMZ cells. ***P<0.001, ##P<0.01 vs HBE cells.

表1 circ_EPHB4表达与临床病理特征的相关性

Tab.1 Correlation between circ_EPHB4 expression and clinicopathological characteristics of glioma patients

Characteristic	Expression of circ_EPHB4
Characteristic	All	Lower expression (n=25)	Higher exprssion (n=25)	P
Gender
Male	37	15	22	0.288
Female	13	10	3
Age(year)
≤60	42	22	20	0.701
>60	8	3	5
Pathological grade
I-II	32	13	19	0.139
III-IV	18	12	6
Tumor size (cm)
<4	28	19	9	0.009
≥4	22	6	16
Preoperative Karnofsky Performance Status (KPS) score
<70	30	10	20	0.008
≥70	20	15	5
Number of tumors
Single	26	17	9	0.065
Multiple	24	8	16

图2 Circ_EPHB4抑制细胞克隆形成,增强TMZ耐药胶质瘤细胞凋亡和TMZ敏感性

Fig.2 Circ_EPHB4 knockdown inhibits cell clone formation and enhances apoptosis and TMZ sensitivity of TMZ-resistant glioma cells. A: qRT-PCR for detecting circ_EPHB4 expression after transfection with si-circ_EPHB4 (**P<0.01). B: CCK-8 assay for assessing viability of TMZ-resistant glioma cells after transfection with si-circ_EPHB4 and treatment with TMZ for 24 h (*P<0.05, **P<0.01 vs si-NC). C: Clone formation assay of cells after transfection with si-circ_EPHB4. D: Flow cytometry for analyzing cell apoptosis after transfection with si-circ_EPHB4. E: Western blotting for detecting expressions of PCNA, MRP1, Bax and P-gp in cells transfected with si-circ_EPHB4.

图3 TMZ耐药胶质瘤细胞Circ_EPHB4与miR-424-5p的相互作用

Fig.3 Interaction between circ_EPHB4 and miR-424-5p in TMZ-resistant glioma cells. A: Comparison of miR-424-5p expression in different tissues. B: Comparison of miR-424-5p expressions in different cells. C: Correlation analysis of miR-424-5p and circ_EPHB4 expression in glioma tissues. D: Detection of miR-424-5p expression by qRT-PCR after transfection with mimics-miR-424-5p. E: Binding sites of miR-424 and circRNA EPHB4. F: RIP assay for detecting expression levels of circ_EPHB4 and miR-424-5p in A172/TMZ cell lysates. G: RIP assay for detecting expression levels of circ_EPHB4 and miR-424-5p in SHG44/TMZ cell lysates. H: Dual-luciferase assay after co-transfection of mimics-miR-424-5p and circ_EPHB4 wt or circ_EPHB4 mut in 293 T cells. *P<0.05, **P<0.01, ***P<0.001; ##P<0.01 vs HBE.

图4 Circ_EPHB4通过调控miR-424-5p表达促进胶质瘤耐药细胞克隆形成、抑制凋亡和降低TMZ敏感性

Fig.4 Circ_EPHB4 promotes clone formation, inhibits apoptosis and reduces TMZ sensitivity of TMZ-resistant glioma cells by regulating the expression of miR-424-5p. A: qRT-PCR for detecting the expression of miR-424-5p in each group of cells. B: CCK-8 assay for assessing cell viability. C: Clone formation assay of the cells. D: Flow cytometry for analyzing cell apoptosis in each group. E: Western blotting for detect the expression levels of PCNA, P-gp, MRP1 and bax in each group. *P<0.05, **P<0.01.

图5 MiR-424-5p靶向Wnt3的3'UTR

Fig.5 MiR-424-5p targets the 3'UTR of Wnt3. A: Comparison of Wnt3 mRNA expression in primary glioma tissues (sensitive/S), recurrent glioma tissues (resistant/R), and healthy brain tissues (normal/N). B: Comparison of Wnt3 mRNA expression levels in HBE, A172, SHG44, A172/TMZ, and SHG44/TMZ cells (##P<0.01 vs HBE cells). C: Comparison of Wnt3 protein expression levels in primary glioma tissues (sensitive/S), recurrent glioma tissues (resistant/R), and healthy brain tissues (normal/N). D: Comparison of Wnt3 protein expression levels in HBE, A172, SHG44, A172/TMZ, and SHG44/TMZ cells. E: Pearson analysis of the correlation between Wnt3 mRNA and miR-424-5p expression in glioma tissues. F: Schematic diagram of the targeting binding sequence between Wnt3 3'-UTR and miR-424-5p. G: Luciferase assay to verify the targeting binding relationship between Wnt3 and miR-424-5p. **P<0.01, ***P<0.001; ##P<0.01 vs HBE.

图6 Wnt3介导miR-424-5p过表达对替莫唑胺耐药胶质瘤细胞克隆形成、细胞凋亡和替莫唑胺体外敏感性的影响

Fig.6 Effects of Wnt3-mediated miR-424-5p overexpression on clone formation, apoptosis and TMZ sensitivity in TMZ-resistant glioma cells. A172/TMZ and SHG44/TMZ cells were transfected with mimics-NC, mimics-miR-424-5p, mimics-miR-424-5p+OE-NC or mimics-miR-424-5p+OE-Wnt3 for 48 h, and the expression levels of Wnt3 mRNA and protein were detected by qRT-PCR and Western blotting (A-D). E: CCK-8 assay for assessing cell viability. F: Clone formation assay of the cells. G: Flow cytometry for analyzing cell apoptosis. H: Expression levels of PCNA, P-gp, MRP1 and bax in the cells detected by Western blotting. *P<0.05, **P<0.01, ***P<0.001.

图7 Circ_EPHB4通过"海绵"吸附miR-424-5p调控Wnt3表达

Fig.7 Circ_EPHB4 regulates the expression of Wnt3 by "sponging" miR-424-5p. The expression of Wnt3 mRNA and protein were detected in A172/TMZ and SHG44/TMZ cells after transfection with si-NC, si-circ_EPHB4, si-circ_EPHB4+inhibitos-NC or si-circ_EPHB4+inhibitos-miR-424-5. A: Expression of Wnt3 mRNA in A172/TMZ cells after transfection. B: Expression of Wnt3 protein in A172/TMZ cells after transfection. C: Expression of Wnt3 mRNA in SHG44/TMZ cells after transfection. D: Expression of Wnt3 protein in SHG44/TMZ cells after transfection. **P<0.01, ***P<0.001.

图8 Circ_EPHB4表达敲低抑制肿瘤生长,促进体内TMZ的敏感性

Fig.8 Knockdown of circ_EPHB4 inhibits tumor growth and enhances TMZ sensitivity of the xenografts in nude mice. A172/TMZ cells transfected with si-NC or si-circ_EPHB4 before subcutaneous implantation in the right flank of nude mice (6 mice in each group). When the tumors reached approximately 100 mm³, PBS (NS) or TMZ (5 mg/kg) was injected intravenously through the tail vein every 3 days. A: Growth curve of the xenografts. B: Comparison of tumor weight. C: qRT-PCR detection of circ_EPHB4, miR-424-5p and Wnt3 mRNA expression levels in the tumors; D: Western blotting for detecting Wnt3 protein expression levels in the tumors. E: Western blotting for detecting PCNA, P-gp, MRP1 and bax expression levels in the tumors. *P<0.05, **P<0.01, ***P<0.001.

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