中国药理学与毒理学杂志2024，Vol.38Issue(7)：517-525,9.DOI:10.3867/j.issn.1000-3002.2024.07.005
G-四链体在糖酵解相关基因中的分布及调控
Distribution and regulation of G-quadruplexes in genes related to glycolysis
刘鹏宇 1蒋兴伟 1马骏 1高峰华 1王哲 1任素萍 1巩家媛 1于群1
作者信息
- 1. 军事医学研究院血液安全保障技术研究北京市重点实验室,北京 100850
- 折叠
摘要
Abstract
OBJECTIVE To investigate the distribution and regulation of G-quadruplex(G4)in enzymes related to glycolysis.METHODS The sequences of the transcription start site(TSS)region upstream of 1500 bp to the 5'-Untranslated region in 200 enzymes and their subtypes in glycolysis were selected for bioinformatics analysis.Related enzymes in glycolysis containing putative G-quadru-plex-forming sequences(PQSs)were identified.Circular Dichroism and Native polyacrylamide gel elec-trophoresis were used to verify the formation of G4.The ExonucleaseⅠhydrolysis assay was used to validate the stability of the formed G4 under 0,0.5,2,8,16,and 32 min.A reporter gene plasmid was constructed by inserting specific fragments of the related enzymes before the luciferase expression sequence.The dual-luciferase reporter assay system validate the expression level of luciferase to assess the impact of G4 on promoter activity.Real-time quantitative PCR was performed to validate the transcriptional regulatory role of G4 by detecting the mRNA levels of luciferase.RESULTS ①Bioin-formatics analysis showed that out of the 200 glycolysis-related enzymes,12 contained PQSs.Based on the analysis of the length and structure of PQSs,aldolase A(ALDOA)and phosphoglycerate mutase 2(PGAM2)proved to be able to form stable G4.② ALDOA and PGAM2 had the maximum positive absorption peak at 260 nm and maximum negative absorption peak at 240 nm.Both of them could form a G4 at the same time.③After digestion with ExonucleaseⅠ,ALDOA and PGAM2 showed no significant hydrolysis and demonstrated the stability of G4 structures.However,both of them could be gradually hydrolyzed after mutations in their PQSs.④ After PQS mutation of ALDOA and PGAM2,the mRNA levels and expression of downstream luciferase of ALDOA were significantly increased(P<0.05,P<0.01),while PGAM2 was significantly decreased(P<0.05,P<0.01).CONCLUSION The gene sequences of glycolysis-related enzymes and their subtypes contain a large number of PQSs.ALDOA and PGAM2 can form stable G4 and perform transcriptional regulatory functions.关键词
G-四链体/潜在G-四链体形成序列/糖酵解/醛缩酶A/磷酸变位酶2/转录调控Key words
G-quadruplexes/putative G-quadruplex-forming sequences/glycolysis/aldolase A/phosphoglycerate mutase 2/transcriptional regulation分类
医药卫生引用本文复制引用
刘鹏宇,蒋兴伟,马骏,高峰华,王哲,任素萍,巩家媛,于群..G-四链体在糖酵解相关基因中的分布及调控[J].中国药理学与毒理学杂志,2024,38(7):517-525,9.
