摘要
目的探索胰岛素样生长因子结合蛋白7(IGFBP7)对胶质瘤细胞替莫唑胺(TMZ)敏感性的影响及作用机制。方法分析基因表达综合数据库(GEO)GSE151680数据集中对TMZ敏感的胶质瘤细胞和TMZ耐药胶质瘤细胞的表达谱测序数据中的IGFBP7 mRNA表达水平;通过药物浓度梯度法诱导建立TMZ耐药胶质瘤细胞(TMZ-R);实时定量PCR(qPCR)、Western印迹检测TMZ-R细胞中IGFBP7的表达水平;通过慢病毒感染在TMZ-R细胞中稳定敲低IGFBP7,在TMZ敏感胶质瘤细胞(TMZ-S)中稳定过表达IGFBP7;采用CCK-8细胞活力实验、细胞凋亡实验、划痕实验、Transwell细胞侵袭实验、克隆形成实验检测细胞活力、凋亡、迁移、侵袭和增殖能力;β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老情况;采用Western印迹检测细胞衰老分子标志物细胞周期依赖性激酶抑制剂1(p21)和肿瘤蛋白p53(p53)、DNA损伤标志物γ-H2A组蛋白家族成员X(γ-H2AX)的表达水平;基于癌症基因组图谱(TCGA)、中国脑胶质瘤基因组图谱计划(CGGA)、基因型-组织表达(GTEx)数据库,分析胶质瘤组织和正常组织中IGFBP7 mRNA表达差异情况及与胶质瘤患者总体生存率的相关性。结果与正常胶质瘤细胞比较,耐药胶质瘤细胞中IGFBP7 mRNA和蛋白水平升高。在TMZ-S细胞中过表达IGFBP7使细胞在TMZ处理下细胞活力升高,凋亡水平降低。在TMZ-R细胞中敲低IGFBP7使细胞在TMZ处理下的细胞活力、增殖、迁移、侵袭能力降低,凋亡和衰老水平升高,p21、p53和γ-H2AX的蛋白表达水平升高。IGFBP7在胶质瘤组织中的mRNA表达水平显著高于正常组织,IGFBP7高表达与胶质瘤患者预后不良显著相关。结论敲低IGFBP7能够促进TMZ诱导的细胞衰老和DNA损伤,提高胶质瘤细胞对TMZ的敏感性。
Objective To investigate the effects of insulin‑like growth factor binding protein 7(IGFBP7)on the sensitivity of glioma cells to temozolomide(TMZ)and the related mechanism.Methods IGFBP7 mRNA expression levels in TMZ‑sensitive(U87 TMZ‑S,U251 TMZ‑S)and TMZ‑resistant(U87 TMZ‑R,U251 TMZ‑R)glioma cells were analyzed using RNA sequencing data from the gene expression omnibus(GEO)dataset GSE151680.TMZ‑resistant U87 and U251 cell lines were established via stepwise dose escalation.IGFBP7 expressions in TMZ‑R cells were detected by quantitative real‑time PCR(qPCR)and Western blotting.IGFBP7 was stably knock‑downed in TMZ‑R cells while IGFBP7 was stably overexpressed in TMZ‑S cells using lentiviral infection.Cell viability,migration,invasion and TMZ sensitivity were assessed using CCK‑8 assay,apoptosis assay,wound healing assay,Transwell invasion assay and colony formation assay respectively.Cellular senescence was detected byβ‑galactosidase(SA‑β‑Gal)staining.The expression levels of senescence molecular markers cyclin‑dependent kinase inhibitor 1(p21)and tumor protein p53(p53),as well as DNA damage markerγ‑H2A histone family member X(γ‑H2AX)were determined by Western blotting.The differences in mRNA expressions of IGFBP7 between glioma tissues and normal tissues as well as the correlations with the overall survival of glioma patients were analyzed using the cancer genome atlas(TCGA),Chinese Glioma Genome Atlas(CGGA),genotype‑tissue expression(GTEx)database.Results Compared to normal glioma cells,IGFBP7 expressions were significantly elevated in TMZ‑R glioma cells.Overexpression of IGFBP7 in TMZ‑S glioma cells enhanced cell viability but suppressed apoptosis following TMZ treatment.The expressions of senescence‑associated marker(p21,p53)and DNA damage marker(γ‑H2AX)were upregulated in these cells.Notably,IGFBP7 expressions were significantly higher in glioma tissues than in normal tissues,and high IGFBP7 expressions were associated with poor prognosis in glioma patients.Conclusion Knockdown of IGFBP7 promotes TMZ‑induced cell senescence and DNA damage,thereby enhancing the sensitivity of gliomas cells to TMZ.
作者
耿晓恩
孙志佳
李江波
周喆
GENG Xiaoen;SUN Zhijia;LI Jiangbo;ZHOU Zhe(Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Air Force Medical Center,Beijing 100142,China)
出处
《军事医学》
2025年第10期728-737,共10页
Military Medical Sciences
