Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)comple...Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.展开更多
目的:通过生物信息学方法,探讨阿尔茨海默病(AD)患者外周血中差异表达的环状RNA (circRNA)的生物学功能。方法:通过基因表达综合数据库(GEO)获取AD相关的数据集GSE186929,筛选AD患者外周血中差异表达的circRNAs,应用Circinteractome和mi...目的:通过生物信息学方法,探讨阿尔茨海默病(AD)患者外周血中差异表达的环状RNA (circRNA)的生物学功能。方法:通过基因表达综合数据库(GEO)获取AD相关的数据集GSE186929,筛选AD患者外周血中差异表达的circRNAs,应用Circinteractome和miRDB数据库来预测circRNA靶向的miRNA,应用Starbase、miWalk、TargetScan8.0在线靶基因预测网站预测靶基因,利用jvenn获得靶基因合集,对差异表达的靶基因进行分析。运用David工具进行基因本体论(GO)和分析和京都基因与基因组百科全书(KEGG)通路分析。通过String在线网站构建蛋白互作(PPI)网络,应用Cytoscape筛选出3个关键基因。结果:共筛选出47个差异表达的circRNAs,其中13个下调,34个上调,选取最具有显著性差异的has_circRNA_0001928进一步预测miRNA,选取数据库中预测效果都较好的miR-142-5p进行靶基因预测,获得63个靶基因。GO和KEGG富集分析显示靶基因参与RNA转录、细胞分裂、基因表达调控等功能以及调节干细胞多能性的信号通路、脂质和动脉粥样硬化信号通路、人类巨细胞病毒感染等信号通路。3个关键基因为PUM2、OTUD4、RANBP2。结论:circRNA及其靶基因可能在AD的发病机制中发挥重要作用,circRNA可能是AD潜在的生物标志物和治疗靶点。Objective: To investigate the biological functions of differentially expressed circular RNAs (circRNAs) in the peripheral blood of Alzheimer’s disease (AD) patients by bioinformatics methods. Methods: AD-related dataset GSE186929 was obtained from Gene Expression Omnibus (GEO), screening for differentially expressed circRNAs in the peripheral blood of AD patients, applying Circinteractome and miRDB databases to predict circRNA-targeted miRNAs, and applying Starbase, miWalk, TargetScan8.0 online target gene prediction website to predict target genes, and used jvenn to obtain target gene ensembles to analyze differentially expressed target genes. Gene ontology (GO) and analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using David tools. Protein Interaction (PPI) network was constructed through String online website and Cytoscape was applied to screen three key genes. Results: A total of 47 differentially expressed circRNAs were screened, of which 13 were down-regulated and 34 were up-regulated. The most significantly different has_circRNA_0001928 was selected for further miRNA prediction, and the miR-142-5p, which had good prediction results in the database, was selected for target gene prediction, resulting in 63 target genes. GO and KEGG enrichment analyses showed that the target genes were involved in the functions of RNA transcription, cell division, and regulation of gene expression as well as the signaling pathways regulating stem cell pluripotency, lipid and atherosclerosis signaling pathways, and human cytomegalovirus infection, etc. The three key genes were PUM2, OTUD4, and RANBP2. Conclusions: CircRNAs and their target genes may play an important role in the pathogenesis of AD, and circRNAs may be potential biomarkers and therapeutic targets for AD.展开更多
肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药...肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药性仍是肺癌死亡率高的原因,其预后不尽如意[2]。环状RNA(CircRNAs)是具有保守、稳定性的一类非编码单链RNA分子[3]。大量的研究表明,CircRNAs在高迁移率族蛋白(high mobility group protein,HMG)家族成员中调节不同的信号通路,在肺癌等多种疾病中发挥关键作用。CircRNAs异常高或低表达可激活或抑制HMG蛋白分子,调节不同生物学过程[4]。随着下一代基因测序技术(next-generation sequencing,NGS)的探索,CircRNAs成为RNA领域肿瘤抑制靶点的研究新热点,为生物医药领域开拓了全新思路,然而其在肺癌中的病理生理机制尚未系统阐明。本文通过总结HMG蛋白家族成员相关CircRNAs在肺癌中的分子作用机制的研究进展,有助于为以CircRNAs为基础的作用靶点及新型抗癌药物的研发提供指导思路,提出潜在有效的治疗策略。展开更多
基金supported by the Guangzhou Science and Technology Plan Project(No.201904010027)the Key Clinical Technology Program of Guangzhou(No.2019ZD18),China。
文摘Background:Circular RNAs(circR NAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circR NA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circR NA.0007127 and its downstream target,microR NA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in HO-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50%and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8in a human myeloid leukemia cell model(75%change)and the level of a 43-kD fragment of CASP8 protein(P small-interfering RNA(siRNA)and the miR-≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.
文摘目的:通过生物信息学方法,探讨阿尔茨海默病(AD)患者外周血中差异表达的环状RNA (circRNA)的生物学功能。方法:通过基因表达综合数据库(GEO)获取AD相关的数据集GSE186929,筛选AD患者外周血中差异表达的circRNAs,应用Circinteractome和miRDB数据库来预测circRNA靶向的miRNA,应用Starbase、miWalk、TargetScan8.0在线靶基因预测网站预测靶基因,利用jvenn获得靶基因合集,对差异表达的靶基因进行分析。运用David工具进行基因本体论(GO)和分析和京都基因与基因组百科全书(KEGG)通路分析。通过String在线网站构建蛋白互作(PPI)网络,应用Cytoscape筛选出3个关键基因。结果:共筛选出47个差异表达的circRNAs,其中13个下调,34个上调,选取最具有显著性差异的has_circRNA_0001928进一步预测miRNA,选取数据库中预测效果都较好的miR-142-5p进行靶基因预测,获得63个靶基因。GO和KEGG富集分析显示靶基因参与RNA转录、细胞分裂、基因表达调控等功能以及调节干细胞多能性的信号通路、脂质和动脉粥样硬化信号通路、人类巨细胞病毒感染等信号通路。3个关键基因为PUM2、OTUD4、RANBP2。结论:circRNA及其靶基因可能在AD的发病机制中发挥重要作用,circRNA可能是AD潜在的生物标志物和治疗靶点。Objective: To investigate the biological functions of differentially expressed circular RNAs (circRNAs) in the peripheral blood of Alzheimer’s disease (AD) patients by bioinformatics methods. Methods: AD-related dataset GSE186929 was obtained from Gene Expression Omnibus (GEO), screening for differentially expressed circRNAs in the peripheral blood of AD patients, applying Circinteractome and miRDB databases to predict circRNA-targeted miRNAs, and applying Starbase, miWalk, TargetScan8.0 online target gene prediction website to predict target genes, and used jvenn to obtain target gene ensembles to analyze differentially expressed target genes. Gene ontology (GO) and analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using David tools. Protein Interaction (PPI) network was constructed through String online website and Cytoscape was applied to screen three key genes. Results: A total of 47 differentially expressed circRNAs were screened, of which 13 were down-regulated and 34 were up-regulated. The most significantly different has_circRNA_0001928 was selected for further miRNA prediction, and the miR-142-5p, which had good prediction results in the database, was selected for target gene prediction, resulting in 63 target genes. GO and KEGG enrichment analyses showed that the target genes were involved in the functions of RNA transcription, cell division, and regulation of gene expression as well as the signaling pathways regulating stem cell pluripotency, lipid and atherosclerosis signaling pathways, and human cytomegalovirus infection, etc. The three key genes were PUM2, OTUD4, and RANBP2. Conclusions: CircRNAs and their target genes may play an important role in the pathogenesis of AD, and circRNAs may be potential biomarkers and therapeutic targets for AD.
文摘肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药性仍是肺癌死亡率高的原因,其预后不尽如意[2]。环状RNA(CircRNAs)是具有保守、稳定性的一类非编码单链RNA分子[3]。大量的研究表明,CircRNAs在高迁移率族蛋白(high mobility group protein,HMG)家族成员中调节不同的信号通路,在肺癌等多种疾病中发挥关键作用。CircRNAs异常高或低表达可激活或抑制HMG蛋白分子,调节不同生物学过程[4]。随着下一代基因测序技术(next-generation sequencing,NGS)的探索,CircRNAs成为RNA领域肿瘤抑制靶点的研究新热点,为生物医药领域开拓了全新思路,然而其在肺癌中的病理生理机制尚未系统阐明。本文通过总结HMG蛋白家族成员相关CircRNAs在肺癌中的分子作用机制的研究进展,有助于为以CircRNAs为基础的作用靶点及新型抗癌药物的研发提供指导思路,提出潜在有效的治疗策略。
