中国癌症防治杂志2025，Vol.17Issue(3)：297-304,8.DOI:10.3969/j.issn.1674-5671.2025.03.06
GCLC启动子甲基化在微囊藻毒素-LR诱导肝细胞恶性转化中的作用及5-氮杂-2'-脱氧胞苷的干预影响
Role of GCLC promoter methylation on microcystin-LR-induced hepatocyte malignant transformation and the interventional effects of 5-aza-2'-deoxycytidine
摘要
Abstract
Objective To investigate the role of glutamate-cysteine ligase catalytic subunit(GCLC)promoter methylation in the malignant transformation of hepatocyte induced by microcystin-LR(MCLR),as well as to assess the intervention effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine(5-aza).Methods A malignant transformation model was established by subjecting WRL68 cells to chronic exposure to 10 nmol/L MCLR until passage 25.Four experimental groups were constituted:the control group,the MCLR-exposed group,the 5-aza intervention group,and the MCLR plus 5-aza intervention group.The methylation status of the GCLC gene promoter was assessed using Agena MassArray mass spectrometry-based nucleic acid analysis technology.Protein expression levels of GCLC and DNA methyltransferases(DNMTs)were determined via Western blot.Cellular proliferation,invasion,and migration capabilities were detected via Colony formation assay,soft agar assay,and Transwell assays,respectively.Additionally,glutamate-cysteine ligase(GCL)activity,glutathione(GSH)content,and 8-hydroxydeoxyguanosine(8-OHdG)levels were quantified using corresponding reagent kits.Results During the MCLR-induced malignant transformation of WRL68 cells,there was a progressive increase in the methylation level of the GCLC promoter,accompanied by a gradual decrease in both mRNA and protein expression levels(all P＜0.05).At passage 25 in the MCLR-exposed group,there was a significant elevation in the protein levels of DNMT-1,DNMT-3a,and DNMT-3b,while GCL activity and GSH content were markedly reduced,and 8-OHdG levels were significantly elevated(all P＜0.05).Intervention with 5-aza at passage 25 in the MCLR-exposed group resulted in a reduction of GCLC promoter methylation levels,significantly upregulating GCLC mRNA and protein expression,enhanced GCL activity and GSH content,and reduced 8-OHdG levels(all P＜0.05).Furthermore,5-aza attenuated the migration,invasion,and cell colony formation capabilities of the MCLR-exposed group(all P＜0.05).Conclusions GCLC promoter methylation contributes to the MCLR-induced malignant transformation of WRL68 cells.5-aza may inhibit this malignant transformation by downregulating DNMTs expression,suppressing hypermethylation of the GCLC promoter,reactivating the expression of the silenced GCLC gene,and restoring its tumor-suppressive function.关键词
谷氨酰半胱氨酸连接酶催化亚基/DNA甲基化/5-氮杂-2'-脱氧胞苷/微囊藻毒素-LR/肝细胞恶性转化Key words
Glutamate-cysteine ligase catalytic subunit/DNA methylation/5-aza-2'-deoxycytidine/Microcystin-LR/Hepatocyte malignant transformation分类
医药卫生引用本文复制引用
聂李云,农清清,李江恒,关斌,黄语莹,陈俞云..GCLC启动子甲基化在微囊藻毒素-LR诱导肝细胞恶性转化中的作用及5-氮杂-2'-脱氧胞苷的干预影响[J].中国癌症防治杂志,2025,17(3):297-304,8.基金项目
国家自然科学基金项目(81960582 （）
82360635) （）
