通用转录因子2I在胶质母细胞瘤替莫唑胺化疗抵抗中的作用

doi:10.13481/j.1671-587X.20240219

摘要/Abstract

摘要：

目的探讨通用转录因子2I（GTF2I）在胶质母细胞瘤（GBM）替莫唑胺化疗抵抗中的作用，并阐明其作用机制。方法基于转录因子预测网站（PROMO网站），生物信息学分析GBM组织中甲基转移酶1（DNMT1）、损伤特异性DNA结合蛋白1（DDB1）、染色盒同源物5（CBX5）和着色性干皮病基因组C（XPC）的共同转录因子，并基于癌症基因组图谱（TCGA）数据库进行DDB1、CBX5、XPC和DNMT1与GTF2I和甲基鸟嘌呤甲基转移酶（MGMT）的相关性分析及生存分析。分别用小干扰序列（siRNA）转染并沉默人脑多形性成胶质细胞瘤T98细胞和人脑胶质瘤LN229细胞中MGMT及GTF2I的基因表达，采用实时荧光定量PCR（RT-qPCR）法检测上述基因的mRNA表达水平。沉默GTF2I基因后，平板克隆形成实验检测肿瘤细胞的集落形成能力，CCK-8法检测细胞对替莫唑胺的敏感性。结果生物信息学分析，GBM组织中DDB1、CBX5、XPC和DNMT1表达水平与GTF2I表达水平呈显著正相关关系（P<0.05），与MGMT表达水平呈负相关关系（P<0.05）；GTF2I表达水平与MGMT表达水平呈显著负相关关系（P<0.05）。剔除未接受替莫唑胺治疗的GBM患者的生存分析，GTF2I高表达的患者总体生存时间降低。沉默MGMT基因后，人脑胶质瘤T98细胞中GTF2I、DDB1、CBX5和XPC mRNA表达水平升高（P<0.001）；沉默GTF2I基因后，人脑胶质瘤LN229细胞中MGMT mRNA表达水平升高（P<0.05），而DDB1、CBX5、XPC和DNMT1 mRNA表达水平明显降低（P<0.05或P<0.001）。平板克隆形成实验，沉默GTF2I基因前后，细胞集落形成能力比较差异无统计学意义（P=0.138）；CCK-8法检测，与对照组比较，观察组细胞活力明显降低（P<0.05）。结论转录因子GTF2I可以调控MGMT、DDB1、CBX5和XPC等关键DNA损伤修复蛋白的mRNA表达，参与GBM细胞替莫唑胺化疗抵抗，可能是GBM潜在的治疗新靶点。

关键词: 胶质母细胞瘤, 替莫唑胺抵抗, 通用转录因子2I, 甲基鸟嘌呤甲基转移酶, 关键DNA损伤修复基因

Abstract:

Objective To discuss the effect of general transcription factor 2I （GTF2I） on the chemotherapeutic resistance to temozolomide in the glioblastoma multiforme （GBM）， and to clarify its mechanism. Methods Bioinformatics analysis was used to identify the common transcription factors for DNA methyltransferase 1 （DNMT1）， damage-specific DNA binding protein 1 （DDB1）， chromobox protein homolog 5 （CBX5）， and xeroderma pigmentosum complementation group C （XPC） in GBM tissue using the transcription factor prediction website （PROMO website）； correlation analysis and survival analysis for DDB1， CBX5， XPC， and DNMT1 with GTF2I and methylguanine methyltransferase （MGMT） were conducted based on The Cancer Genome Atlas （TCGA） Database；small interfering RNAs （siRNAs） were used to transfect and silence the gene expression of MGMT and GTF2I in the human T98 GBM cells and LN229 glioma cells； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of the gene mRNA. After silencing the GTF2I gene， plate clone formation assay was used to detect the colon forming ability of the tumor cells， and CCK-8 assay was used to detect the sensitivity of the cells to temozolomide. Results The bioinformatics analysis results showed that the expression levels of DDB1， CBX5， XPC， and DNMT1 were significantly positively correlated with the expression level of GTF2I in GBM tissue （P<0.05）， and were negatively correlated with the expression level of MGMT （P<0.05）. The expression level of GTF2I was significantly negatively correlated with the expression level of MGMT （P<0.05）. Excluding the GBM patients who did not receive temozolomide treatment， the survival analysis results indicated that the patients with high expression of GTF2I had a decreased overall survival time. After silencing the MGMT gene， the expression levels of GTF2I， DDB1， CBX5， and XPC mRNA in the human brain glioma T98 cells were increased （P<0.001）； after silencing the GTF2I gene， the expression level of MGMT mRNA in the human brain glioma LN229 cells was increased （P<0.05）， while the expression levels of DDB1， CBX5， XPC， and DNMT1 mRNA were significantly decreased （P<0.05 or P<0.001）. The plate clone formation assay results showed that there was no significant difference in the clone formation ability of the cells before and after silencing the GTF2I gene （P=0.138）. The CCK-8 assay results showed that compared with control group，the viability of the cells in observation group was decreased （P<0.05）. Conclusion The transcription factor GTF2I regulates the expressions of key DNA damage repair protein mRNA， including MGMT， DDB1， CBX5， and XPC， and is involved in the chemotherapeutic resistance of the GBM cells to temozolomide；GTF2I may represent a potential new therapeutic target for the GBM.

Key words: Glioblastoma multiforme, Temozolomide resistance, Universal transcription factor 2I, Methylguanine methyltransferase, Key DNA damage repair genes

中图分类号:

R739.41

周建国,姜红建,朱其辉,张耿强,邓琪琳,齐玲,李凯舒,于洪泉. 通用转录因子2I在胶质母细胞瘤替莫唑胺化疗抵抗中的作用[J]. 吉林大学学报(医学版), 2024, 50(2): 457-464.

Jianguo ZHOU,Hongjian JIANG,Qihui ZHU,Gengqiang ZHANG,Qilin DENG,Ling QI,Kaishu LI,Hongquan YU. Effect of general transcription factor 2I on temozolomide chemotherapy resistance of glioblastoma[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 457-464.

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Primer	Sequence (5′－3′)
GTF2I-1	Forward Reverse	GGCCAAGUCCAAAGCCGAATT UUCGGCUUUGGACUUGGCCTT
GTF2I-2	Forward Reverse	GGUGGUCGUGUGAUGGUAATT UUACCAUCACACGACCACCTT
GTF2I-3	Forward Reverse	CCACAGAAGAUUCUGGCAUTT AUGCCAGAAUCUUCUGUGGTI
MGMT-1	Forward Reverse	GCAGGGUCUGCACGAAAUATT UAUUUCGUGCAGACCCUGCTT
MGMT-2	Forward Reverse	GCAAUGAGAGGCAAUCCUGTT CAGGAUUGCCUCUCAUUGCTT
MGMT-3	Forward Reverse	GCUGCUGAAGGUUGUGAAATT UUUCACAACCUUCAGCAGCTT

Primer	Sequence (5′－3′)
GTF2I	Forward Reverse	GCATGTAGGTGGTCGTGTGA CAGTTTCAGAAGGGCCTTGA
MGMT	Forward Reverse	GAAGCTGGAGCTGTCTGGTT CGGGGAACTCTTCGATAGCC
CBX5	Forward Reverse	TAGACAGGCGCGTGGTTAAG AGCCCCGAGCGATATCATTG
DDB1	Forward Reverse	CAAAGCACGATTGTGTGCCA TGAGCTTCACAAGCTGGGAG
XPC	Forward Reverse	TGCCTATTGGCTGTGTCCAG AGACCTTTGGCCAGCAACTT
DNMT1	Forward Reverse	AGGAGGGCTACCTGGCTAAA GCTTAGCCTCTCCATCGGAC

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