目的开发一种基于滚环扩增(rolling circle amplification,RCA)技术的高灵敏度外周血G-四链体(guanine quadruplex,G4)检测方法,建立ATRX突变型脑胶质瘤的无创分子诊断体系,为临床早期筛查提供新型液体活检策略。方法采用shRNA慢病毒感...目的开发一种基于滚环扩增(rolling circle amplification,RCA)技术的高灵敏度外周血G-四链体(guanine quadruplex,G4)检测方法,建立ATRX突变型脑胶质瘤的无创分子诊断体系,为临床早期筛查提供新型液体活检策略。方法采用shRNA慢病毒感染技术建立ATRX稳定敲低的原代脑胶质瘤细胞株(ATRX KD),通过硫黄素T(thioflavin T,ThT)荧光光谱法检测细胞上清液中G4的形成水平。构建ATRX敲低的原位脑胶质瘤小鼠模型,采用RCA变温扩增技术检测模型组与对照组外周血循环游离DNA(cfDNA)中G4的丰度,并对RCA变温扩增技术的程序进行优化。收集临床确诊的ATRX突变型和野生型胶质瘤患者,以及健康志愿者的血清样本,提取cfDNA后采用优化的RCA方案进行G4定量检测,并再次对检测程序进行优化。结果体外实验显示,ATRX敲低可显著促进G4结构的形成,ATRX KD组细胞上清液中ThT荧光强度较野生型显著增加。建立RCA变温扩增方案检测ATRX突变型胶质瘤小鼠模型外周血cfDNA中G4结构的实验条件:以50 ng cfDNA为起始模板、进行10个循环扩增。临床样本验证显示,优化后的RCA变温扩增方案较传统恒温扩增效率提升,能够有效区分ATRX突变型与野生型胶质瘤患者及健康人群。结论成功建立了基于RCA变温扩增的外周血G4高灵敏度检测方案,该技术首次实现ATRX突变状态的液体活检分子分型,为胶质瘤早期筛查提供具有临床应用前景的无创诊断方法。展开更多
Objective:ATRX is a multifunctional protein that is tightly regulated by and implicated in transcriptional regulation and chromatin remodeling.Numerous studies have shown that genetic alterations in ATRX play a signif...Objective:ATRX is a multifunctional protein that is tightly regulated by and implicated in transcriptional regulation and chromatin remodeling.Numerous studies have shown that genetic alterations in ATRX play a significant role in gliomas.This study aims to further determine the relationship between ATRX and glioma prognosis and identify possible mechanisms for exploring the biological significance of ATRX using large data sets.Methods:We used The Cancer Genome Atlas(TCGA)database and 130 immunohistochemical results to confirm the difference in ATRX mutations in high-and low-grade gliomas.An online analysis of the TCGA glioma datasets using the cBioPortal platform was performed to study the relationship between ATRX mutations and IDH1,TP53,CDKN2 A and CDKN2 B mutations in the corresponding TCGA glioma dataset.In combination with clinical pathology data,the biological significance of the relationships were analyzed.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses and annotations of all adjacent genes in the network were performedin the Database for Annotation,Visualization and Integrated Discovery(DAVID)and R language.A protein-protein interaction(PPI)network was constructed,and the interactions of all adjacent nodes were analyzed by the String database and using Cytoscape software.Results:In the selected TCGA glioma datasets,a total of 2,228 patients were queried,21%of whom had ATRX alterations,which co-occurred frequently with TP53 and IDH1 mutations.ATRX alterations are associated with multiple critical molecular events,which results in a significantly improved overall survival(OS)rate.In low-grade gliomas,ATRX mutations are significantly associated with multiple important molecular events,such as ZNF274 and FDXR at mRNA and protein levels.A functional cluster analysis revealed that these genes played a role in chromatin binding and P53,and a link was observed between ATRX and IDH1 and TP53 in the interaction network.ATRX and TP53 are important nodes in the network and have potential links with the blood oxygen imbalance.Conclusions:ATRX mutations have clinical implications for the molecular diagnosis of gliomas and can provide diagnostic and prognostic information for gliomas.ATRX is expected to serve as a new therapeutic target.展开更多
文摘目的开发一种基于滚环扩增(rolling circle amplification,RCA)技术的高灵敏度外周血G-四链体(guanine quadruplex,G4)检测方法,建立ATRX突变型脑胶质瘤的无创分子诊断体系,为临床早期筛查提供新型液体活检策略。方法采用shRNA慢病毒感染技术建立ATRX稳定敲低的原代脑胶质瘤细胞株(ATRX KD),通过硫黄素T(thioflavin T,ThT)荧光光谱法检测细胞上清液中G4的形成水平。构建ATRX敲低的原位脑胶质瘤小鼠模型,采用RCA变温扩增技术检测模型组与对照组外周血循环游离DNA(cfDNA)中G4的丰度,并对RCA变温扩增技术的程序进行优化。收集临床确诊的ATRX突变型和野生型胶质瘤患者,以及健康志愿者的血清样本,提取cfDNA后采用优化的RCA方案进行G4定量检测,并再次对检测程序进行优化。结果体外实验显示,ATRX敲低可显著促进G4结构的形成,ATRX KD组细胞上清液中ThT荧光强度较野生型显著增加。建立RCA变温扩增方案检测ATRX突变型胶质瘤小鼠模型外周血cfDNA中G4结构的实验条件:以50 ng cfDNA为起始模板、进行10个循环扩增。临床样本验证显示,优化后的RCA变温扩增方案较传统恒温扩增效率提升,能够有效区分ATRX突变型与野生型胶质瘤患者及健康人群。结论成功建立了基于RCA变温扩增的外周血G4高灵敏度检测方案,该技术首次实现ATRX突变状态的液体活检分子分型,为胶质瘤早期筛查提供具有临床应用前景的无创诊断方法。
基金support of the Special Construction Innovation Funded Project for Community in Beijing,Tianjin and Hebei of China(Grant No.18247792D)the Hebei Basic Research Cooperation Project(Grant No.H2018201306)
文摘Objective:ATRX is a multifunctional protein that is tightly regulated by and implicated in transcriptional regulation and chromatin remodeling.Numerous studies have shown that genetic alterations in ATRX play a significant role in gliomas.This study aims to further determine the relationship between ATRX and glioma prognosis and identify possible mechanisms for exploring the biological significance of ATRX using large data sets.Methods:We used The Cancer Genome Atlas(TCGA)database and 130 immunohistochemical results to confirm the difference in ATRX mutations in high-and low-grade gliomas.An online analysis of the TCGA glioma datasets using the cBioPortal platform was performed to study the relationship between ATRX mutations and IDH1,TP53,CDKN2 A and CDKN2 B mutations in the corresponding TCGA glioma dataset.In combination with clinical pathology data,the biological significance of the relationships were analyzed.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses and annotations of all adjacent genes in the network were performedin the Database for Annotation,Visualization and Integrated Discovery(DAVID)and R language.A protein-protein interaction(PPI)network was constructed,and the interactions of all adjacent nodes were analyzed by the String database and using Cytoscape software.Results:In the selected TCGA glioma datasets,a total of 2,228 patients were queried,21%of whom had ATRX alterations,which co-occurred frequently with TP53 and IDH1 mutations.ATRX alterations are associated with multiple critical molecular events,which results in a significantly improved overall survival(OS)rate.In low-grade gliomas,ATRX mutations are significantly associated with multiple important molecular events,such as ZNF274 and FDXR at mRNA and protein levels.A functional cluster analysis revealed that these genes played a role in chromatin binding and P53,and a link was observed between ATRX and IDH1 and TP53 in the interaction network.ATRX and TP53 are important nodes in the network and have potential links with the blood oxygen imbalance.Conclusions:ATRX mutations have clinical implications for the molecular diagnosis of gliomas and can provide diagnostic and prognostic information for gliomas.ATRX is expected to serve as a new therapeutic target.
