冠状动脉粥样硬化患者单个核细胞关键RNA结合蛋白基因的筛选及验证

Identification and validation of key RNA-binding protein genes in mononuclear cells of patients with coronary atherosclerosis

目的筛选冠状动脉粥样硬化患者外周血单个核细胞关键RNA结合蛋白基因,并对其进行验证。方法①冠状动脉粥样硬化患者单个核细胞关键RNA结合蛋白基因筛选选择冠状动脉粥样硬化患者5例(观察组)、健康志愿者5例(对照组),采用转录组测序技术检测外周血单个核细胞RNA转录组,运用R软件“edgeR”包筛选P<0.05、|logFC|>1、错误发现率<0.05的单个核细胞差异表达基因。从RNA结合蛋白数据库中下载人单个核细胞的RNA结合蛋白相关基因资料,与外周血单个核细胞差异表达基因取交集后获得冠状动脉粥样硬化单个核细胞RNA结合蛋白相关差异表达基因。运用TopHat2、ABLas软件筛选冠状动脉粥样硬化患者单个核细胞差异表达RNA结合蛋白相关基因的可变剪接事件。筛选相对表达量最高的单个核细胞RNA结合蛋白相关差异表达基因为关键RNA结合蛋白基因。②冠状动脉粥样硬化患者单个核细胞关键RNA结合蛋白基因验证另选取冠状动脉粥样硬化患者10例(一组)、健康志愿者10例(二组),抽取外周静脉血后分离外周血单个核细胞,采用qRT-PCR法检测2组RNA结合蛋白关键基因。从GEO数据库(GSE40231、GSE43292、GSE100927、GSE27034)数据集中,检索冠状动脉粥样硬化患者、健康志愿者外周血单个核细胞RNA结合蛋白关键基因表达资料。结果冠状动脉粥样硬化患者单个核细胞的差异RNA结合蛋白相关基因有:过氧化物酶体增殖物激活受体γ共激活因子1α、锌指RNA结合蛋白2、SAM域、SH3域和核定位信号1(SAMSN1)、肝配蛋白A型受体2、RNA结合基序蛋白24、多种剪接形式的RNA结合蛋白2、SH3和多个锚蛋白重复结构域1。与对照组相比,观察组患者单个核细胞RNA结合蛋白相关基因的可变剪接事件多(P<0.05)。关键RNA结合蛋白基因为SAMSN1。与二组相比,一组患者外周血单个核细胞SAMSN1相对表达量升高(P<0.05)。GEO数据库数据结果显示,相比于健康志愿者,冠状动脉粥样硬化患者单个核细胞单个核细胞SAMSN1相对表达量升高(P<0.05)。结论冠状动脉粥样硬化患者单个核细胞关键RNA结合蛋白基因为SAMSN1基因。相比于健康志愿者,冠状动脉粥样硬化患者单个核细胞SAMSN1基因表达升高,可变剪接事件增多。SAMSN1基因可能通过调控RNA结合蛋白相关基因的可变剪接事件,参与冠状动脉粥样硬化的发生发展。

Objective To screen the key differentially expressed RNA-binding proteins(RBPs)genes in peripheral blood mononuclear cells of patients with coronary atherosclerosis and to validate them.Methods①Screening of key differentially expressed RBPs genes in mononuclear cells of patients with coronary atherosclerosis:We selected 5 cases of patients with coronary atherosclerosis(observation group)and 5 cases of healthy volunteers(control group),used transcriptome sequencing technology to detect the RNA transcriptome of peripheral blood mononuclear cells,and used the"edgeR"package in R software to screen all differentially expressed genes of mononuclear cells with P<0.05,|logFC|>1,false discovery rate<0.05.We download human mononuclear cell RBP-related genes from the RBP database,intersected with differentially expressed genes of peripheral blood mononuclear cells to obtain RBP-related differentially expressed genes of mononuclear cells in patients with coronary atherosclerosis.We used TopHat2,ABLas software to screen for alternative splicing events of differentially expressed RBP-related genes in mononuclear cells of patients with coronary atherosclerosis.We screened the key RBP gene with the highest relative expression among differentially expressed RBP-related genes in mononuclear cells.②Validation of key RBP genes in mononuclear cells of patients with coronary atherosclerosis:We selected another 10 cases of patients with coronary atherosclerosis(group one)and 10 cases of healthy volunteers(group two),drew peripheral venous blood and isolated peripheral blood mononuclear cells,and used qRT-PCR method to detect the key RBP genes of the two groups.We retrieved the expression data of key RBP genes in peripheral blood mononuclear cells of patients with coronary atherosclerosis and healthy volunteers from GEO database datasets(GSE40231,GSE43292,GSE100927,GSE27034).Results The differentially expressed RBP-related genes in the mononuclear cells of patients with coronary atherosclerosis included peroxisome proliferator-activated receptor gamma coactivator 1 alpha(PPARGC1A),zinc finger RNA-binding protein 2(ZFR2),SAM domain,SH3 domain,and nuclear localization signal 1(SAMSN1),Ephrin type-A receptor 2(EPHA2),RNA-binding motif protein 24(RBM24),RNA-binding protein with multiple splicing 2(RBPMS2),and SH3 and multiple ankyrin repeat domains 1(SHANK1).Compared with the control group,there were more alternative splicing events of RBP-related genes in the mononuclear cells of the observation group(all P<0.05).The key RBP-related gene was SAMSN1.Compared with the group two,the group one had higher relative expression level of SAMSN1 in peripheral blood mononuclear cells of patients(P<0.05).Data from the GEO database showed that the relative expression level of SAMSN1 in mononuclear cells was higher in patients with coronary atherosclerosis than in healthy volunteers(P<0.05).Conclusions The key RBP gene in mononuclear cells of patients with coronary atherosclerosis is the SAMSN1 gene.In patients with coronary atherosclerosis,the expression of the SAMSN1 gene in mononuclear cells is elevated,and the alternative splicing events increase.The SAMSN1 gene may participate in the development and progression of coronary atherosclerosis by regulating alternative splicing events of RBP-related genes.