脓肿分枝杆菌感染THP-1巨噬细胞自噬相关分子的识别

Recognition of Autophagy-related Molecules of MAB-infected THP-1 Macrophages

为了探索THP-1巨噬细胞在脓肿分枝杆菌(Mycobacterium abscessus,MAB)感染后,与自噬相关的关键分子及其介导的生物学过程,从GEO和GeneCards数据库中挖掘MAB感染后THP-1巨噬细胞的自噬相关基因;利用DAVID在线工具对筛选的差异自噬相关基因进行基因本体论(GO)、京都基因和基因组数据库(KEGG)富集分析;采用实时荧光定量PCR(qRT-PCR)方法验证筛选的关键基因;将关键基因与自噬标志分子MAP1LC3A进行Pearson相关性分析。生物信息学分析显示,与自噬密切相关的差异表达基因共11个,其中前5个是BNIP3(Bcl-2/adenovirus E1 B interacting protein 3)、BAMBI(BMP and activin membrane bound inhibitor)、MAP1LC3A(microtubule associated protein 1 light chain 3)、DYSF(Dysferlin)和MYOZ1(Myozenin1);功能预测显示,11个差异表达基因主要和自噬等过程相关;qRT-PCR验证结果显示,BNIP3、MAP1LC3A和MYOZ1在两种表型MAB感染后的THP-1巨噬细胞中的变化与转录组测序(RNA-seq)结果一致,且BNIP3在RNA-seq和qRT-PCR结果中差异最显著(P<0.05);Pearson相关性分析显示,BNIP3与MAP1LC3A的相关性最强(P<0.05)。综上所述,BNIP3是MAB感染THP-1巨噬细胞后与自噬相关性最强的自噬相关分子,这可能为MAB感染巨噬细胞后的自噬过程的分子机制研究提供重要的启示。

To explore the key autophagy-related molecules and the biological processes mediated by THP-1macrophages after Mycobacterium abscesses(MAB)infection,the autophagy-related genes of MAB-infected THP-1 macrophages were collected from GEO and GeneCards databases.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of the selected genes were performed by the DAVID online tool.Real-time quantitative PCR(qRT-PCR)was used to verify the screened key genes.The Pearson correlations between the key genes and the autophagy marker molecule were analyzed.Bioinformatics analysis indicated that there were 11 genes closely related to autophagy,and the top five with the largest expression differences were Bcl-2/adenovirus E1 B interacting protein 3(BNIP3),BMP and activin membrane bound inhibitor(BAMBI),microtubule associated protein 1 light chain 3(MAP1LC3A),Dysferlin(DYSF)and Myozenin1(MYOZ1).Function prediction showed that,after MAB infection of macrophages,11 differentially expressed genes were mainly related to processes such as protein binding and autophagy.qRT-PCR verification results showed that the changes of BNIP3,MAP1LC3A and MYOZ1 in THP-1 macrophages infected with two phenotypes of MAB were consistent with the results of transcriptome sequencing(RNA-seq).The difference between RNA-seq and RT-PCR results of BNIP3 was the most significant(P<0.05).Pearson correlation analysis showed that BNIP3 had the strongest correlation with MAP1LC3A(P<0.05).Therefore,BNIP3proved to be the most autophagy-related molecule after MAB infection of THP-1 macrophages.The study may provide an important clue for exploring molecular mechanisms of autophagy in MAB-infected macrophages.