circ_EPHB4协同YTHDF3通过N6-甲基腺苷依赖性稳定Wnt3促进胶质瘤进展

doi:10.12122/j.issn.1673-4254.2025.11.04

摘要/Abstract

摘要：

目的探讨环状RNA circ_EPHB4在胶质瘤中的促癌作用及其与N6-甲基腺苷（m⁶A）阅读蛋白YTHDF3协同调控Wnt3信号通路的分子机制。方法通过芯片分析筛选胶质瘤组织中差异表达的circRNA，利用划痕愈合实验、Transwell侵袭实验及皮下成瘤模型，检测circ_EPHB4对胶质瘤细胞迁移、侵袭、上皮-间质转化（EMT）及体内成瘤能力的影响。采用RNA免疫沉淀、RNA稳定性实验及基因过表达/敲低技术，验证circ_EPHB4与YTHDF3对Wnt3 表达的协同调控作用。结果 circ_EPHB4在胶质瘤组织中较癌旁组织高表达2.3倍（|log2FC|=1.2，P<0.01），在胶质瘤细胞株U373中表达量达正常细胞的2.5倍（P<0.001）。过表达circ_EPHB4可使胶质瘤细胞划痕愈合率提升至（75.2±6.3）%，较对照组增加78.6%（P<0.01）；侵袭细胞数增至215±23个/视野，为对照组2.4倍（P<0.001），并促进EMT标志物N-cadherin和Vimentin的表达（P<0.001）。体内实验显示，过表达组肿瘤体积21 d达1200±150 mm³，较对照组增加140%（P<0.001）；肺转移灶数6.3±1.2个，为对照组4.2倍（P<0.01）。circ_EPHB4 通过上调Wnt3表达（P<0.01）发挥促癌作用，而YTHDF3 以m⁶A依赖性方式延长Wnt3 mRNA半衰期至11.5±1.2 h，较对照组增加40%（P<0.01）。二者协同调控时，同时敲低可使Wnt3 mRNA表达降至单独敲低组的47%（P<0.001）。结论 circ_EPHB4与YTHDF3通过共同靶向Wnt3信号通路促进胶质瘤进展，靶向该协同轴可能为治疗提供新策略。

关键词: circ_EPHB4, N6-甲基腺苷, YTHDF3, Wnt3, 胶质瘤

Abstract:

Objective To investigate the oncogenic role of circular RNA circ_EPHB4 in glioma and its molecular mechanism. Methods Microarray analysis was performed to identify the differentially expressed circRNAs in glioma tissues. The effects of circ_EPHB4 on glioma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and tumorigenicity in vivo were assessed using scratch wound healing assay, Transwell invasion assay and nude mouse models bearing subcutaneous tumors. RNA immunoprecipitation (RIP), RNA stability assays, and gene overexpression and silencing techniques were employed to validate the synergistic regulatory effect of circ_EPHB4 and the N6-methyladenosine (m⁶A) reader protein YTHDF3 on Wnt3 expression. Results Circ_EPHB4 was significantly overexpressed by 2.3 folds (|log2FC|=1.2, P<0.01) in glioma tissues compared to the adjacent tissues, and by 2.5 folds in glioma cell line U373 compared to normal cells (P<0.001). Overexpression of circ_EPHB4 significantly enhanced migration and invasion of glioma cells, and promoted the expressions of EMT markers N-cadherin and vimentin. In the tumor-bearing mouse models, the tumor volume in circ_EPHB4 overexpression group was significantly greater than that in the control group, and the lung metastatic foci increased by 4.2 folds. Overexpression of circ_EPHB4 promoted oncogenesis by upregulating Wnt3 expression, while YTHDF3 extended the half-life of Wnt3 mRNA in an m⁶A-dependent manner. Simultaneous knockdown of circ_EPHB4 and YTHDF3 resulted in an obvious reduction of Wnt3 mRNA expression by up to 47% compared to its level following knocking down either circ_EPHB4 or YTHDF3 alone. Conclusion Circ_EPHB4 and YTHDF3 promote glioma progression by jointly targeting the Wnt3 signaling pathway, which may provide a new therapeutic strategy for gliomas.

Key words: circ_EPHB4, m⁶A, YTHDF3, Wnt3, glioma

金晨, 刘景平, 刘博, 费喜云, 廖宇翔. circ_EPHB4协同YTHDF3通过N6-甲基腺苷依赖性稳定Wnt3促进胶质瘤进展[J]. 南方医科大学学报, 2025, 45(11): 2320-2329.

Chen JIN, Jingping LIU, Bo LIU, Xiyun FEI, Yuxiang LIAO. circ_EPHB4 synergizes with YTHDF3 to promote glioma progression via m⁶A-dependent stabilization of Wnt3[J]. Journal of Southern Medical University, 2025, 45(11): 2320-2329.

图/表 6

表1 目的基因及内参引物序列

Tab.1 Primer sequences for RT-qPCR of the target and housekeeping genes

Gene	Forward Primer (5'-3')	Reverse Primer (5'-3')
circ_EPHB4	CAGAGAGCGCATCCGCAATTT	AAAGAAGGTGCGCCCACGGTT
Zeb1	AACAGTGAGCGACCTTCATT	AGAGCGCAGAGAAGGAGAGTT
GAPDH	AGAGCGCAGAGAAGGAGAUTT	AAAGAGCGAGAGAAGGAGAT

图1 circ_EPHB4在胶质瘤中表达上调

Fig.1 circ_EPHB4 is upregulated in glioma. A: Differential RNA expression analysis shows increased circ_EPHB4 expression in glioma. B: qRT-PCR confirms increased expression of circ_EPHB4 in glioma (***P<0.001). C: circ_EPHB4 expressions are significantly higher in glioma cell lines U251, A172, SHG44, LN229, and U373 than in normal human brain astrocyte HEB cells. *P<0.05, **P<0.01, ***P<0.001 vs HEB.

图2 circ_EPHB4促进胶质瘤细胞迁移、侵袭和EMT

Fig.2 circ_EPHB4 promotes glioma cell migration, invasion, and EMT. A: qRT-PCR of circ_EPHB4 expression in glioma cells after transfection with circ_EPHB4-overexpressing plasmids or circ_EPHB4 siRNA (**P<0.01). B: Scratch wound healing assay for evaluating glioma cell migration ability following circ_EPHB4 overexpression or knockdown (scale bar=100 μm). C: Transwell invasion assay for assessing glioma cell invasion ability after transfection (scale bar=50 μm). D: Western blotting of EMT-related proteins (E-cadherin, N-cadherin and vimentin) in glioma cells following circ_EPHB4 overexpression or knockdown. Data are presented as Mean±SD from 3 independent experiments. *P<0.05 vs siRNA NC; #P<0.05 vs pcDNA3.1 NC.

图3 circ_EPHB4促进胶质瘤细胞体内成瘤及转移

Fig.3 circ_EPHB4 promotes tumorigenesis and metastasis of glioma cells in nude mice. A: Number of pulmonary metastatic nodules in each group. B: Subcutaneous tumor volume at different time points in each group. C: Subcutaneous tumor weight in each group (*P<0.05, **P<0.01, ***P<0.001). D: qRT-PCR analysis of mRNA expressions of E-cadherin, N-cadherin, vimentin) in subcutaneous tumors. E: Immunohistochemicalanalysis of protein expressions of E-cadherin, N-cadherin, and vimentin in subcutaneous tumors from each group. **P<0.01, ***P<0.001 vs siRNA NC group; ##P<0.01, ### P<0.001 vs pcDNA3.1 NC group.

图4 circ_EPHB4通过上调Wnt3表达促进胶质瘤细胞转移

Fig.4 circ_EPHB4 promotes glioma cell metastasis by upregulating Wnt3 expression. A: Overexpression of circ_EPHB4 enhances Wnt3 expression, and circ_EPHB4 knockdown suppresses Wnt3 expression. B: Scratch wound healing assay for evaluating the effect of Wnt3 knockdown on glioma cell migration following circ_EPHB4 overexpression (scale bar=50 μm). C: Transwell invasion assay for assessing the impact of Wnt3 knockdown on invasion ability of circ_EPHB4-overexpressing glioma cells (scale bar=50 μm). D: Western blotting for evaluating the effects of Wnt3 knockdown on expressions of E-cadherin, N-cadherin and vimentin in circ_EPHB4-overexpressing glioma cells.

图5 YTHDF3通过m6A依赖性方式增强Wnt3 mRNA的稳定性

Fig.5 YTHDF3 enhances Wnt3 mRNA stability in an m6A-dependent manner. A: Direct interaction between YTHDF3 and Wnt3 mRNA in U3T3 and SHG44 cells. B: Effect of YTHDF3 knockdown on Wnt3 expression. C: Positive correlation between YTHDF3 and Wnt3 expression in the StarBase database. D: Effect of circ_EPHB4 on the interaction between YTHDF3 and Wnt3 RNA. E: Effect of combined knockdown of circ_EPHB4 and YTHDF3 on Wnt3 mRNA expression. *P<0.05, **P<0.01, ***P<0.001.

参考文献 31

[1]	Ludwig K, Kornblum HI. Molecular markers in glioma[J]. J Neurooncol, 2017, 134(3): 505-12. doi：10.1007/s11060-017-2379-y
[2]	翟贝, 魏灯, 王萍. tRNA修饰在胶质瘤中的研究进展[J]. 中国肿瘤临床, 2024,51(11): 567-71.
[3]	贾金曦, 姚春旭, 刘畅, 等. 脑胶质瘤患者术后远期预后影响因素分析[J]. 实用癌症杂志, 2024, 39(3): 514-7.
[4]	Liu ZX, Li LM, Sun HL, et al. Link between m6A modification and cancers[J]. Front Bioeng Biotechnol, 2018, 6: 89. doi：10.3389/fbioe.2018.00089
[5]	Zhang G, Cheng C, Wang X, et al. N6-Methyladenosine methylation modification in breast cancer: current insights[J]. J Transl Med, 2024, 22(1): 971. doi：10.1186/s12967-024-05771-x
[6]	Rui Y, Zhang H, Yu K, et al. N⁶-methyladenosine regulates Cilia elongation in cancer cells by modulating HDAC6 expression[J]. Adv Sci: Weinh, 2025, 12(2): e2408488. doi：10.1002/advs.202408488
[7]	Chen XH, Guo KX, Li J, et al. Regulations of m6A and other RNA modifications and their roles in cancer[J]. Front Med, 2024, 18(4): 622-48. doi：10.1007/s11684-024-1064-8
[8]	Qin L, Zeng X, Qiu X, et al. The role of N6-methyladenosine modification in tumor angiogenesis[J]. Front Oncol, 2024, 14: 1467850. doi：10.3389/fonc.2024.1467850
[9]	Saluja S, Ganguly S, Singh J, et al. Aberrant overexpression of m6A writer and reader genes in pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL)[J]. Transl Oncol, 2025, 56: 102403. doi：10.1016/j.tranon.2025.102403
[10]	Zhao Y, Feng F, Guo QH, et al. Role of succinate dehydrogenase deficiency and oncometabolites in gastrointestinal stromal tumors[J]. World J Gastroenterol, 2020, 26(34): 5074-89. doi：10.3748/wjg.v26.i34.5074
[11]	Li J, Han Y, Zhang HM, et al. The m6A demethylase FTO promotes the growth of lung cancer cells by regulating the m6A level of USP7 mRNA[J]. Biochem Biophys Res Commun, 2019, 512(3): 479-85. doi：10.1016/j.bbrc.2019.03.093
[12]	Dong J, Zeng Z, Huang Y, et al. Challenges and opportunities for circRNA identification and delivery[J]. Crit Rev Biochem Mol Biol, 2023, 58(1): 19-35. doi：10.1080/10409238.2023.2185764
[13]	Chen X, Lu Y. Circular RNA: biosynthesis in vitro [J]. Front Bioeng Biotechnol, 2021, 9: 787881. doi：10.3389/fbioe.2021.787881
[14]	Patop IL, Kadener S. circRNAs in cancer[J]. Curr Opin Genet Dev, 2018, 48: 121-7. doi：10.1016/j.gde.2017.11.007
[15]	Hanan M, Soreq H, Kadener S. CircRNAs in the brain[J]. RNA Biol, 2017, 14(8): 1028-34. doi：10.1080/15476286.2016.1255398
[16]	Chen M, Wei L, Law CT, et al. RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS₂ [J]. Hepatology, 2018, 67(6): 2254-70. doi：10.1002/hep.29683
[17]	Yin J, Ding F, Cheng Z, et al. METTL3-mediated m6A modification of LINC00839 maintains glioma stem cells and radiation resistance by activating Wnt/β-catenin signaling[J]. Cell Death Dis, 2023, 14(7): 417. doi：10.1038/s41419-023-05933-7
[18]	Xiao L, Li X, Mu Z, et al. FTO inhibition enhances the anti-tumor effect of temozolomide by targeting MYC-miR-155/23a cluster-MXI1 feedback circuit in glioma. Cancer Res. 2020. 80(18): canres.0132.2020. doi：10.1158/0008-5472.can-20-0132
[19]	Zheng W, Li J, Zhou X, et al. The lncRNA XIST promotes proliferation, migration and invasion of gastric cancer cells by targeting miR-337[J]. Arab J Gastroenterol, 2020, 21(3): 199-206. doi：10.1016/j.ajg.2020.07.010
[20]	Patop IL, Wüst S, Kadener S. Past, present, and future of circRNAs[J]. EMBO J, 2019, 38(16): e100836. doi：10.15252/embj.2018100836
[21]	Yu T, Wang Y, Fan Y, et al. CircRNAs in cancer metabolism: a review[J]. J Hematol Oncol, 2019, 12(1): 90. doi：10.1186/s13045-019-0776-8
[22]	Yang R, Cui J. Advances and applications of RNA vaccines in tumor treatment[J]. Mol Cancer, 2024, 23(1): 226. doi：10.1186/s12943-024-02141-5
[23]	Yi Q, Feng JG, Lan WW, et al. CircRNA and lncRNA-encoded peptide in diseases, an update review[J]. Mol Cancer, 2024, 23(1): 214. doi：10.1186/s12943-024-02131-7
[24]	Zhang S, Zhang P, Wu A, et al. Downregulated M6A modification and expression of circRNA_103239 promoted the progression of glioma by regulating the miR-182-5p/MTSS1 signalling pathway[J]. J Cancer, 2023, 14(18): 3508-20. doi：10.7150/jca.85320
[25]	Dai YH, Zhao SH, Chen HL, et al. RNA methylation and breast cancer: insights into m6A, m7G and m5C[J]. Mol Biol Rep, 2024, 52(1): 27. doi：10.1007/s11033-024-10138-y
[26]	Lin ZY, Wan AH, Sun L, et al. N6-methyladenosine demethylase FTO enhances chemo-resistance in colorectal cancer through SIVA1-mediated apoptosis[J]. Mol Ther, 2023, 31(2): 517-34. doi：10.1016/j.ymthe.2022.10.012
[27]	Zhang C, Huang S, Zhuang H, et al. YTHDF2 promotes the liver cancer stem cell phenotype and cancer metastasis by regulating OCT4 expression via m6A RNA methylation[J]. Oncogene, 2020, 39(23): 4507-18. doi：10.1038/s41388-020-1303-7
[28]	Chang H, Yang J, Wang Q, et al. Role of N6-methyladenosine modification in pathogenesis of ischemic stroke[J]. Expert Rev Mol Diagn, 2022, 22(3): 295-303. doi：10.1080/14737159.2022.2049246
[29]	Li L, Zhang T, Xiao M, et al. Brain macrophage senescence in glioma[J]. Semin Cancer Biol, 2024, 104/105: 46-60. doi：10.1016/j.semcancer.2024.07.005
[30]	Davidson CL, Vengoji R, Jain M, et al. Biological, diagnostic and therapeutic implications of exosomes in glioma[J]. Cancer Lett, 2024, 582: 216592. doi：10.1016/j.canlet.2023.216592
[31]	Yin JL, Liu G, Zhang Y, et al. Gender differences in gliomas: From epidemiological trends to changes at the hormonal and molecular levels[J]. Cancer Lett, 2024, 598: 217114. doi：10.1016/j.canlet.2024.217114