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作 者:刘鹏宇 蒋兴伟 马骏 高峰华 王哲 任素萍 巩家媛 于群 LIU Pengyu;JIANG Xingwei;MA Jun;GAO Fenghua;WANG Zhe;REN Suping;GONG Jiayuan;YU Qun(Beijing Key Laboratory of Blood Safety Supply Technologies,Academy of Military Medical Sciences,Beijing 100850,China)
机构地区:[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
出 处:《中国药理学与毒理学杂志》2024年第7期517-525,共9页Chinese Journal of Pharmacology and Toxicology
摘 要:目的探讨G-四链体(G4)在糖酵解相关基因中的分布及调控。方法选取200个糖酵解相关基因转录起始位点上游1500 bp至5′非翻译区的序列进行生物信息学分析,初步确定含有潜在G-四链体形成序列(PQS)的相关基因;圆二色谱法和非变性聚丙烯酰胺凝胶电泳法检测G4形成;外切酶Ⅰ(ExoⅠ)水解实验在0,0.5,2,8,16和32 min检测G4稳定性;构建将相关基因的特定片段插入到荧光素酶表达序列之前的报告基因质粒,双荧光素酶报告基因系统检测荧光素酶的表达水平进而判断G4对启动子活性的影响;实时荧光定量PCR检测荧光素酶的mRNA水平进一步验证G4的转录调控作用。结果①生物信息学分析表明,200个糖酵解相关基因中有12个基因含有PQS,进一步结合PQS长度及结构分析,醛缩酶A(ALDOA)和磷酸变位酶2(PGAM2)的PQS理论上可形成稳定G4。②ALDOA和PGAM2均在260 nm处有最大正吸收峰,在240 nm处有最大负吸收峰,符合平行结构G4的特征构象,同时二者的PQS可形成G4。③ExoⅠ消化后,ALDOA和PGAM2无明显水解,证明G4具有稳定性,同时二者的PQS突变后均可逐渐被水解。④ALDOA突变后荧光素酶的表达水平和mRNA水平显著升高(P<0.01);PGAM2突变后荧光素酶的表达水平和mRNA水平显著降低(P<0.01)。结论糖酵解相关基因的序列中存在大量PQS,其中ALDOA和PGAM2的PQS可形成稳定G4,并具有转录调控作用。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-quadruplex-forming sequences(PQSs)were identified.Circular Dichroism and Native polyacrylamide gel electrophoresis 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.RESULTS①Bioinformatics 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 G4.②ALDOA 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 Exonuclease Ⅰ,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.④After 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 tra
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