CircCpsf6在N2a细胞中对细胞活力的抑制作用及其结合的蛋白功能富集分析  

Inhibitory effect of CircCpsf6 on cell viability in N2a cells and functional enrichment analyses of its binding proteins

作　　者：铁昌锐 万峻[1] TIE Chang-rui;WAN Jun(Biomedical Research Institute,Shenzhen-Peking University-The Hong Kong University of Science and Technology Medical Center,Shenzhen,Guangdong Province 518036,China)

机构地区：[1]深圳北京大学香港科技大学医学中心生物医学研究所,广东深圳518036

出　　处：《解剖学研究》2021年第3期193-198,205,共7页Anatomy Research

基　　金：国家自然科学基金项目(82071586)。

摘　　要：目的探讨CircCpsf6在N2a细胞中对细胞活力的影响,同时对CircCpsf6结合蛋白进行功能富集分析,预测CircCpsf6影响细胞活力的分子机制。方法在N2a细胞中转染CircCpsf6的siRNA或过表达质粒,对CircCpsf6进行敲减或过表达,用CCK-8法检测细胞活力。在CircCpsf6的接口处设计特异性探针,进行下拉实验,即CircCpsf6的反义纯化实验。经过磁珠吸附,蛋白的洗脱、纯化,蛋白的质谱检测,得到CircCpsf6可能结合的蛋白。利用在线网站STRING对这些蛋白之间可能存在的相互作用进行预测,同时利用在线网站DAVID进行蛋白功能富集分析。结果在N2a细胞中,CircCpsf6能够被有效的敲减或过表达(P<0.01),敲减CircCpsf6后,细胞活力上升(P<0.01);过表达CircCpsf6后,细胞活力下降(P<0.05)。经过CircCpsf6的反义纯化实验,发现CircCpsf6可能与385个蛋白结合。GO分析和KEGG富集分析提示CircCpsf6可能通过结合蛋白影响细胞的增殖或者凋亡,从而影响细胞活力。结论CircCpsf6能够抑制N2a细胞的细胞活力。CircCpsf6可能通过结合G6pdx、Pcx、Csl影响细胞增殖,通过结合2210010C04Rik、Try10、Gm2663影响细胞凋亡。Objective To explore the effect of CircCpsf6 on cell viability in N2a cells.At the same time,functional enrichment analyses of CircCpsf6-bound proteins were performed to predict the molecular mechanism of CircCpsf6 affecting cell viability.Methods CircCpsf6 was knocked down or overexpressed by the transfection of siRNA or overexpression plasmid in N2a cells.CCK-8 assay was used to detect cell viability.A specific probe was designed at the junction of CircCpsf6 to conduct the pull-down experiment,namely the antisense purification experiment of CircCpsf6.After magnetic bead adsorption,protein elution,purification,and protein mass spectrometry detection,CircCpsf6 could be bound to the protein.The online website STRING was used to predict the possible interactions between these proteins,and the online website DAVID was used to perform protein function enrichment analysis.Results CircCpsf6 could be effectively knocked down or overexpressed in N2a cells(P<0.01).Cell viability was increased after the knockdown of CircCpsf6(P<0.01),and cell viability was decreased after the overexpression of CircCpsf6(P<0.05).After the antisense purification experiment of CircCpsf6,it was found that CircCpsf6 could bind to 385 proteins.GO enrichment analysis and KEGG enrichment analysis suggested that CircCpsf6 may affect cell proliferation or apoptosis through binding proteins,thus affecting cell viability.Conclusion CircCpsf6 inhibited the cell viability of N2a cells.CircCpsf6 may affect cell proliferation by binding to G6pdx,Pcx,Csl,and affect cell apoptosis by binding to 2210010C04Rik,Try10,Gm2663.

关 键 词：小鼠脑神经瘤细胞 N2a细胞 环状RNA 环状裂解和多聚腺苷酸化特异因子6 细胞活力 功能富集分析 

分 类 号：R739.41[医药卫生—肿瘤]