WTAP通过调节BMI1 mRNA的m6A修饰促进胶质瘤细胞增殖和有氧糖酵解

doi:10.19401/j.cnki.1007-3639.2023.07.003

摘要/Abstract

摘要：

背景与目的：甲基转移酶Wilms瘤1-相关蛋白（Wilms’ tumor 1-associating protein，WTAP）被证实在肿瘤中发挥重要作用，本研究旨在从N6-甲基腺苷（N6-methyladenosine，m6A）修饰的角度探讨WTAP在胶质瘤发生、发展过程中的作用及其机制。方法：通过芯片筛选胶质瘤中差异表达的m6A调节因子，采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）评估WTAP/B细胞特异性莫洛尼氏鼠白血病病毒整合位点1（B cell-specific Moloney murine leukemia virus integration site 1，BMI1）在胶质瘤组织和细胞系中的表达。借助四甲基偶氮唑盐（methyl thiazoyl terazolium，MTT）探究WTAP/BMI1对胶质瘤细胞增殖的作用。采用细胞外酸化率（extracellular acidification rate，ECAR）试剂盒、葡萄糖消耗及乳酸生成检测试剂盒评估胶质瘤细胞有氧糖酵解情况。结果：相对于正常组织，WTAP、BMI1在胶质瘤组织中表达上调。此外，相对于HEB细胞中的WTAP（1.01±0.13）和BMI1（1.02±0.11）表达，U251细胞中的WTAP（2.38±0.17）、BMI1（2.25±0.14）表达增强（P均 <0.05）。敲减WTAP在体外能抑制细胞增殖和糖酵解能力，而过表达WTAP则显示相反效果。过表达WTAP对胶质瘤细胞增殖、糖酵解的作用能被敲减BMI1部分挽救（P均<0.05）。在机制上，WTAP促进BMI1的m6A甲基化修饰，使BMI1 mRNA的稳定性增强（P均<0.05）。结论：WTAP能以m6A甲基化依赖方式促进BMI1表达，从而促进胶质瘤细胞增殖和有氧糖酵解，靶向WTAP的分子治疗手段可能为改善胶质瘤治疗效果提供新方向。

关键词: 胶质瘤, N6-甲基腺苷修饰, 有氧糖酵解, 甲基转移酶

Abstract:

Background and purpose: Methyltransferase Wilms’ tumor 1-associating protein (WTAP) has been proven to play an important role in the development of tumors. This study aimed to explore the influence of WTAP on the occurrence and development of glioma from the perspective of N6-methyladenosine (m6A) modification. Methods: The differentially expressed m6A regulators in glioma were screened from GEO database. The expression of WTAP/B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) in glioma tissue and cell lines was evaluated by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. Methyl thiazoyl terazolium (MTT) was used to explore the effects of WTAP/BMI1 on glioma cell proliferation. Extracellular acidification rate (ECAR) kits and glucose consumption and lactate production detection kits were utilized to detect aerobic glycolysis of glioma cells. Results: Compared with normal tissues, WTQP and BMI1 expressions were upregulated in glioma tissues. Otherwise, compared with WTAP (1.01±0.13) and BMI1 (1.02±0.11) expressions in HEB cells, WTAP (2.38±0.17) and BMI1 (2.25±0.14) expressions in U251 cells were increased (P<0.05). Downregulation of WTAP inhibited cell proliferation and glycolytic potential in vitro, while overexpression of WTAP showed opposite effects. The effects of WTAP overexpression on glioma cell proliferation and glycolysis were partially rescued by BMI1 inhibition (P<0.05). WTAP promoted m6A methylation modification of BMI1 and enhanced the stability of BMI1 mRNA (P<0.05). Conclusion: WTAP upregulates the level of BMI1 in a m6A methylation modification dependent manner, subsequently promotes glioma cell proliferation and aerobic glycolysis. Molecular therapy targeting WTAP may provide a new method for the treatment of glioma.

Key words: Glioma, N6-methyladenosine modification, Aerobic glycolysis, Methyltransferase

中图分类号:

R739.41

刁新峰, 李新茂, 候亮, 魏志玄. WTAP通过调节BMI1 mRNA的m6A修饰促进胶质瘤细胞增殖和有氧糖酵解[J]. 中国癌症杂志, 2023, 33(7): 655-663.

DIAO Xinfeng, LI Xinmao, HOU Liang, WEI Zhixuan. WTAP promotes proliferation and aerobic glycolysis via regulating m6A modification of BMI1 mRNA[J]. China Oncology, 2023, 33(7): 655-663.

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Gene	Forward primer (5‘-3‘)	Reverse primer (5‘-3‘)
WTAP	GCGACTAGCAACCAAGGAAC	CATTTTGGGCTTGTTCCAGT
BMI1	CCAGGGCTTTTCAAAAATGA	CCGATCCAATCTGTTCTGGT
GAPDH	CGACCACTTTGTCAAGCTCA	AGGGGTCTACATGGCAACTG

Clinical data	Case n (n=47)	WTAP expression		P value
Clinical data	Case n (n=47)	High expression n	Low expression n	P value
Gender				0.920
Male	25	14	11
Female	22	12	10
Age/year				0.927
≤45	16	9	7
>45	31	17	14
Tumor size/cm				0.474
<3	20	9	11
≥3	27	15	12
WHO grade				0.014 5
Ⅰ-Ⅱ	31	11	20
Ⅲ-Ⅳ	16	12	4

Gene	RF classifier	SVM classifier
MYB	0.65	0.96
APC	0.70	0.98
TP53	0.70	0.79
EGFR	0.80	0.94
BMI1	0.80	0.97
PPM1D	0.65	0.95
ALK	0.80	0.96
NTRK1	0.75	0.59
HLA-A	0.85	0.20