METHODS: Microsomal glutathione S-transferase 1 (MGST1) expression in glioma samples was ensured through GAPIA database, qRT-PCR, western blotting assay and immunohistochemistry. The interaction between zinc finger protein 384 (ZNF384) and MGST1 promoter was analyzed through UCSC and JASPAR databases and further verified by ChIP and luciferase reporter assay. Cell viability and IC50 value of temozolomide (TMZ) was measured by CCK-8 assay. The production of MDA, GSH and ROS and the level of Fe2+ were determined using the corresponding kit.
RESULTS: MGST1 expression was increased in clinical glioma tissues and glioma cells. MGST1 expression was increased but ferroptosis was suppressed in TMZ-resistant cells when contrasted to parent cells. MGST1 silencing downregulated IC50 value of TMZ and cell viability but facilitated ferroptosis in TMZ-resistant cells and parent glioma cells. Moreover, our data indicated that ZNF384 interacted with MGST1 promoter and facilitated MGST1 expression. ZNF384 was also increased expression in TMZ-resistant cells, and showed a positive correlation with MGST1 expression in clinical level. ZNF384 decreasing enhanced the sensitivity of resistant cells to TMZ, while the effect of ZNF384 could be reversed by overexpression of MGST1.
CONCLUSIONS: MGST1 transcription is regulated by transcription factor ZNF384 in TMZ-resistant cells. ZNF384 confers the resistance of glioma cells to TMZ through inhibition of ferroptosis by positively regulating MGST1 expression. The current study may provide some new understand to the mechanism of TMZ resistance in glioma.
方法:通过GAPIA数据库确保胶质瘤样本中微粒体谷胱甘肽S-转移酶1(MGST1)的表达,qRT-PCR,蛋白质印迹法和免疫组织化学。通过UCSC和JASPAR数据库分析锌指蛋白384(ZNF384)与MGST1启动子之间的相互作用,并通过ChIP和荧光素酶报告基因测定进一步验证。通过CCK-8测定测量替莫唑胺(TMZ)的细胞活力和IC50值。MDA的产生,使用相应的试剂盒测定GSH和ROS以及Fe2+的水平。
结果:MGST1在临床胶质瘤组织和胶质瘤细胞中表达增加。与亲本细胞相比,TMZ抗性细胞中的MGST1表达增加,但铁性凋亡受到抑制。MGST1沉默下调TMZ的IC50值和细胞活力,但促进TMZ抗性细胞和亲本神经胶质瘤细胞的铁凋亡。此外,我们的数据表明ZNF384与MGST1启动子相互作用并促进MGST1表达。ZNF384在TMZ抗性细胞中的表达也增加,临床水平与MGST1表达呈正相关。ZNF384的降低增强了耐药细胞对TMZ的敏感性,而过表达MGST1可以逆转ZNF384的作用。
结论:在TMZ抗性细胞中,MGST1转录受转录因子ZNF384调控。ZNF384通过正向调节MGST1表达抑制铁凋亡而赋予神经胶质瘤细胞对TMZ的抗性。目前的研究可能为胶质瘤中TMZ耐药的机制提供一些新的认识。