主动脉夹层细胞焦亡相关miRNAs筛选及功能分析

Identification and functional analysis of pyroptosis-related miRNAs in aortic dissection

目的分析并验证急性主动脉夹层(acute aortic dissection,AAD)细胞焦亡相关miRNAs的功能。方法下载GEO数据库中基于miRNA芯片的微阵列数据集,筛选差异表达的miRNAs,通过miRWalk数据库预测靶基因。以“pyroptosis”为关键词在PubMed数据库中搜索与细胞焦亡相关基因(pyroptosis-related genes,PRGs),韦恩图取PRGs和差异miRNAs预测的靶基因交集为AAD的PRGs,并进行GO、KEGG富集分析。采用cytoHubba筛选AAD关键PRGs,进而确定AAD细胞焦亡相关miRNAs。收集性别、年龄匹配的AAD患者与健康人群的主动脉组织,Western blotting及RT-qPCR验证关键基因及miRNAs。结果共筛选出46个AAD差异表达的miRNAs,通过韦恩图得到49个AAD的PRGs。GO富集分析显示,这些基因在半胱氨酸内肽酶调控的凋亡中起重要作用。KEGG富集分析发现,这些基因富集于沙门菌感染、坏死性凋亡和Nod样受体信号通路。cytoHubba筛选的AAD关键PRGs为半胱氨酸天冬氨酸酶-1(cysteine aspartase-1,Caspase-1)、白细胞介素(interleukin,IL)-1β和肿瘤坏死因子(tumor necrosis factor,TNF),进而获得12个AAD细胞焦亡相关miRNAs。分别获取AAD和健康人群主动脉组织,各6例,其中AAD组男5例、女1例,平均年龄(48.70±6.35)岁;健康对照组男4例、女2例,平均年龄(45.30±4.58)岁。两组性别、年龄、吸烟史、高血压病史、糖尿病史和冠心病史差异均无统计学意义(P>0.05)。Western blotting及RT-qPCR结果显示,与健康主动脉相比,AAD患者主动脉组织中Caspase-1表达上调,对应的miRNAs为miR-198、miR-3202和miR-514b-5p,表达均下调。结论AAD关键PRGs为Caspase-1、IL-1β和TNF,其中Caspase-1表达上调,3个对应的miRNAs表达下调,为AAD细胞焦亡的分子机制及靶向治疗提供了新思路。

Objective To screen pyroptosis-related miRNAs of acute aortic dissection(AAD)from the GEO database,and analyze and verify their functions.Methods The microarray data set based on the miRNA chip in the GEO database was downloaded,the differentially expressed miRNAs were screened,and the target genes were predicted by the miRWalk database.Pyroptosis-related genes(PRGs)were searched in the PubMed database with"pyroptosis"as the keyword,and the intersection of PRGs and differential miRNAs predicting target genes were taken as AAD PRGs by Venn diagram.GO and KEGG enrichment analyses were performed.CytoHubba was used to screen the critical AAD PRGs and then the AAD pyroptosis-related miRNAs were identified.Aortic tissues were collected from gender-and age-matched AAD patients and healthy people,and the critical PRGs and miRNAs were verified by Western blotting and RT-qPCR.Results A total of 46 AAD differentially expressed miRNAs were screened,and 49 AAD PRGs were obtained by Venn diagram.GO enrichment analysis showed that the genes played a vital role in apoptosis regulated by cysteine endopeptidases.KEGG analysis showed that the genes enriched in Salmonella infection,necroptosis,and Nod-like receptor signaling pathways.CytoHubba screened the critical AAD PRGs such as cysteine aspartase-1(Caspase-1),tumor necrosis factor(IL)-1β,and tumor necrosis factor(TNF),then obtained 12 AAD pyroptosis-related miRNAs.Aortic tissues were collected from 6 AAD patients and 6 healthy people.There were 5 males and 1 females in the AAD group with an average age of 48.70±6.35 years,and 4 males and 2 females in the healty control group with an average age of 45.30±4.58 years.There was no statistical difference between the two groups in terms of gender,age,smoking history,hypertension,diabetes,or coronary heart disease(P>0.05).Western blotting and RT-qPCR results showed that Caspase-1 was up-regulated in the AAD patients'aortic tissues compared with the healthy aorta,and the corresponding miRNAs were miR-198,miR-3202,and miR-514b-5p,which were all down-regulated.Conclusion Through bioinformatics analysis and verification,the critical AAD PRGs are Caspase-1,IL-1β,and TNF,and Caspase-1 is up-regulated and 3 corresponding pyroptosis-related miRNAs are down-regulated,which provides new ideas for the molecular mechanism and targeted therapy of AAD cell pyroptosis.