ALKBH5通过调节CPNE1 m6A甲基化影响非小细胞肺癌顺铂化疗敏感性

doi:10.19586/j.2095-2341.2025.0164

生物技术进展 ›› 2026, Vol. 16 ›› Issue (2): 412-421.DOI: 10.19586/j.2095-2341.2025.0164

• 研究论文 • 上一篇

ALKBH5通过调节CPNE1 m⁶A甲基化影响非小细胞肺癌顺铂化疗敏感性

刘鸿飞¹(), 赵文², 杨慧英³()

^1.国家电网公司北京电力医院，北京 100073
^2.开封市中心医院医学影像科，河南开封 475000
^3.北京市羊坊店医院消毒供应室，北京 100038

收稿日期:2025-11-18 接受日期:2026-01-05 出版日期:2026-03-25 发布日期:2026-04-27
通讯作者: 杨慧英
作者简介:刘鸿飞 E-mail: 187347309@qq.com；
基金资助:
河南省开封市科技攻关项目(2303079)

ALKBH5 Affects Cisplatin Chemosensitivity in Non-small Cell Lung Cancer by Regulating CPNE1 m⁶A Methylation

Hongfei LIU¹(), Wen ZHAO², Huiying YANG³()

^1.Beijing Electric Power Hospital，State Grid Corporation，Beijing 100073，China
^2.Department of Medical Imaging，Kaifeng Central Hospital，Henan Kaifeng 475000，China
^3.Disinfection and Supply Department of Beijing Yangfangdian Hospital，Beijing 100038，China

Received:2025-11-18 Accepted:2026-01-05 Online:2026-03-25 Published:2026-04-27
Contact: Huiying YANG

摘要/Abstract

摘要：

旨在探讨N6-甲基腺苷（N6-methyladenosine, m⁶A）去甲基化酶烷化修复同源物5（alkB homolog 5, ALKBH5）通过调控钙磷脂结合蛋白1（Copine 1, CPNE1）mRNA的m⁶A甲基化对非小细胞肺癌（non-small cell lung cancer，NSCLC）顺铂（cisplatin, DDP）化疗敏感性的影响及其机制。采用qRT-PCR和Western blot检测ALKBH5和CPNE1在肺癌细胞系中的表达水平；通过基于序列的RNA腺苷甲基化位点预测工具（sequence-based RNA adenosine methylation site predictor，SRAMP）预测CPNE1的m⁶A甲基化位点，Me-RIP检测其甲基化水平，RNA免疫共沉淀（RNA immunoprecipitation，RIP）实验验证ALKBH5与CPNE1 mRNA的结合。分别转染OE-CPNE1、si-CPNE1和si-ALKBH5至肺癌细胞H157，CCK-8检测细胞活力并计算IC₅₀，克隆形成实验评估细胞增殖水平，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase dUTP nick end labeling，TUNEL）染色检测凋亡。在si-CPNE1转染的细胞中加入PI3K/AKT激活剂740Y-P，分析CPNE1对PI3K/AKT通路的作用。结果发现，NSCLC细胞中ALKBH5和CPNE1的表达显著高于正常肺上皮细胞（P<0.05）。敲低ALKBH5降低了CPNE1的表达并提升了m⁶A甲基化水平（P<0.05）。RIP实验证实ALKBH5与CPNE1 mRNA存在直接结合。抑制ALKBH5或CPNE1可显著增强顺铂对肺癌细胞的杀伤作用，表现为细胞活力降低、增殖抑制和凋亡增加（P<0.05）。此外，PI3K/AKT激活剂740Y-P可恢复因CPNE1敲低所致的通路抑制及化疗耐药表型。由此表明，ALKBH5可通过m⁶A依赖的方式上调CPNE1表达，激活PI3K/AKT通路，从而降低NSCLC细胞对顺铂的化疗敏感性。

关键词: 非小细胞肺癌, m⁶A甲基化, ALKBH5, CPNE1, PI3K/AKT, 化疗敏感性

Abstract:

This study aimed to investigate the effect and mechanism of the N6-methyladenosine （m⁶A） demethylase alkB homolog 5 （ALKBH5） on cisplatin （DDP） chemosensitivity in non-small cell lung cancer （NSCLC） by regulating the m⁶A methylation of CPNE1 （Copine 1） mRNA. The expression levels of ALKBH5 and CPNE1 in lung cancer cell lines were detected by qRT-PCR and Western blot. The sequence-based RNA adenosine methylation site predictor （SRAMP） was used to predict the m⁶A methylation sites of CPNE1， Me-RIP assay was performed to detect its methylation level， and RNA immunoprecipitation （RIP） assay was conducted to verify the binding between ALKBH5 and CPNE1 mRNA. Lung cancer H157 cells were transfected with OE-CPNE1， si-CPNE1， and si-ALKBH5， respectively. Cell viability was detected by CCK-8 assay to calculate the IC₅₀ value， colony formation assay was used to evaluate cell proliferation， and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was employed to detect cell apoptosis. The PI3K/AKT activator 740Y-P was added to si-CPNE1-transfected cells to analyze the role of CPNE1 in the PI3K/AKT pathway. The results showed that the expressions of ALKBH5 and CPNE1 in NSCLC cells were significantly higher than those in normal lung epithelial cells （P<0.05）. Knockdown of ALKBH5 decreased the expression of CPNE1 and increased its m⁶A methylation level （P<0.05）. The RIP assay confirmed a direct binding between ALKBH5 and CPNE1 mRNA. Inhibition of ALKBH5 or CPNE1 significantly enhanced the cytotoxic effect of cisplatin on lung cancer cells， which was manifested by decreased cell viability， inhibited proliferation， and increased apoptosis （P<0.05）. In addition， the PI3K/AKT activator 740Y-P could rescue the pathway inhibition and chemoresistance phenotype induced by CPNE1 knockdown. These findings indicated that ALKBH5 upregulated CPNE1 expression in an m⁶A-dependent manner and activated the PI3K/AKT pathway， thereby reducing the cisplatin chemosensitivity of NSCLC cells.

Key words: non-small cell lung cancer, m⁶A methylation, ALKBH5, CPNE1, P13/AKT, chemosensitivity

中图分类号:

Q279

刘鸿飞, 赵文, 杨慧英. ALKBH5通过调节CPNE1 m⁶A甲基化影响非小细胞肺癌顺铂化疗敏感性[J]. 生物技术进展, 2026, 16(2): 412-421.

Hongfei LIU, Wen ZHAO, Huiying YANG. ALKBH5 Affects Cisplatin Chemosensitivity in Non-small Cell Lung Cancer by Regulating CPNE1 m⁶A Methylation[J]. Current Biotechnology, 2026, 16(2): 412-421.

图/表 5

图1 ALKBH5在非小细胞肺癌细胞中的表达A：Western blot检测人正常肺上皮细胞（BEAS-2B）及3种非小细胞肺癌细胞（NCI-H358、H157、NCI-H1299）中ALKBH5的蛋白表达水平；B：ALKBH5蛋白表达的定量分析柱状图。*表示在P<0.05水平组间差异具有统计学意义。

Fig. 1 Expression of ALKBH5 in NSCLC cells

图2 ALKBH5对CPNE1 m6A甲基化修饰的影响A：Western blot检测人正常肺上皮细胞和非小细胞肺癌细胞中CPNE1蛋白表达；B：CPNE1蛋白表达的定量分析柱状图；C：MeRIP-qPCR检测各组细胞中CPNE1 mRNA的m6A甲基化水平；D：SRAMP预测CPNE1的m6A甲基化修饰位点；E：qRT-PCR检测各组H157细胞中ALKBH5 mRNA表达；F：Western blot检测各组H157细胞中ALKBH5蛋白表达；G： Western blot检测各组H157细胞中CPNE1蛋白表达；H：qRT-PCR检测各组H157细胞中CPNE1 mRNA表达；I：MeRIP-qPCR各组H157细胞中CPNE1 mRNA的m6A甲基化水平；J：RIP实验检测ALKBH5与CPNE1的相互作用。*表示在P<0.05水平组间差异具有统计学意义。

Fig. 2 Effect of ALKBH5 on m6A methylation modification of CPNE1

图3 CPNE1对肺癌细胞顺铂化疗敏感性的影响A：qRT-PCR分析各组细胞中CPNE1 mRNA表达水平；B：Western blot检测各组细胞中CPNE1蛋白表达水平；C：CCK-8法测定不同浓度顺铂处理下各组细胞的活力并计算IC50；D：CCK-8法检测10 μg·mL-1顺铂处理下各组细胞的活力；E：克隆形成实验评估各组细胞增殖能力的影响；F：TUNEL染色分析细胞凋亡情况。*表示在P<0.05水平组间差异具有统计学意义。

Fig. 3 Effect of CPNE1 on cisplatin chemosensitivity in lung cancer cells

图4 ALKBH5对肺癌细胞对顺铂的化疗敏感性的影响A：qRT-PCR分析各组细胞中CPNE1 mRNA表达水平；B：Western blot检测各组细胞中CPNE1和ALKBH5蛋白表达水平；C：qRT-PCR分析各组细胞中ALKBH5 mRNA表达水平；D：CCK-8法测定不同浓度顺铂处理下各组细胞的活力并计算IC50；E：CCK-8法检测10 μg·mL-1顺铂处理下各组细胞的活力；F：克隆形成实验评估各组细胞增殖能力；G：TUNEL染色分析细胞凋亡情况。*表示在P<0.05水平组间差异具有统计学意义。

Fig. 4 Effects of ALKBH5 on cisplatin chemosensitivity in lung cancer cells

图5 CPNE1通过激活PI3K/AKT通路降低肺癌细胞化疗敏感性A：Western blott检测蛋白表达水平；B：CCK-8法检测10 μg·mL-1顺铂处理下各组细胞的活力；C：克隆形成实验评估各组细胞增殖能力；D：TUNEL染色分析细胞凋亡。*表示在P<0.05水平组间差异具有统计学意义。

Fig. 5 CPNE1 reduces chemosensitivity in lung cancer cells by activating the PI3K/AKT pathway

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ALKBH5通过调节CPNE1 m⁶A甲基化影响非小细胞肺癌顺铂化疗敏感性

ALKBH5 Affects Cisplatin Chemosensitivity in Non-small Cell Lung Cancer by Regulating CPNE1 m⁶A Methylation

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