基于WGCNA分析筛选c-MYC/Bcl-2重排弥漫大B细胞淋巴瘤的关键基因

Screening key genes of diffuse large B-cell lymphoma with c-MYC/Bcl-2 translocation based on WGCNA analysis

目的:筛选c-MYC/Bcl-2重排的弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)潜在的高风险致病基因及致病通路,为此类淋巴瘤的发病机制提供理论依据。方法:从基因表达数据库(GEO)下载GSE44164及GSE43677数据集,选择c-MYC/Bcl-2重排的DLBCL数据为实验组、生发中心B细胞为对照组。采用R程序语言的limma包、WGCNA包及clusterProfiler包对获得的mRNA转录组数据进行差异表达分析、基因共表达变化分析、GO功能富集分析与KEGG信号通路富集分析。使用STRING数据库对差异表达基因(DEGs)进行蛋白互相作用网络分析,并使用Cytoscape软件内置的插件(包括CytoHubba及CytoNca插件)筛选核心基因。采用第二代高通量测序技术对IM-9细胞系及DOHH-2细胞系进行mRNA转录组测序,使用Read count值将核心基因进行配对样本t检验,筛选P值具有统计学意义的基因为疾病的核心基因。挑选部分关键基因使用Western Blot技术在蛋白层面进行验证。结果:差异分析共得到835个DEGs,WGCNA获得一个与c-MYC/Bcl-2重排的DLBCL高度相关的模块(turquoise模块,cor=0.86,P值<0.05)。GO富集分析结果显示生物学进程BP共富集到1 437条,细胞组分CC富集到123条,分子功能相关MF富集到147条;KEGG富集到72条相关通路,主要与ECM受体相互作用、B细胞受体信号通路及PI3K-Akt信号通路等相关。STRING数据库中PPI网络共得到284个相互关联作用的蛋白质,通过Cytoscape软件进一步筛选,再通过二代高通量测序及蛋白验证得出COL1A1、COL3A1、COL1A2、MMP2、COL5A1、COL5A2、COL4A2、TIMP1、MMP9、POSTN、BGN、DCN、LUM为此类淋巴瘤的核心基因,影响此类淋巴瘤细胞生存、侵袭和迁移等。结论:使用生物信息学及基础实验相结合的方法探究与c-MYC/Bcl-2重排的DLBCL淋巴瘤细胞致病或侵袭迁移等相关的关键基因,为更深入地探索此类淋巴瘤发生发展机制及寻找相关靶向药物提供理论依据。

Objective:To identify the potential high-risk genes and pathogenic pathways in DLBCL with c-MYC/Bcl-2 translocation,and to establish a theoretical foundation for understanding the pathogenesis of this specific lymphoma subtype.Methods:GSE44164 and GSE43677 datasets were acquired from the Gene Expression Omnibus(GEO) database.The experimental group consisted of DLBCL cases with c-MYC/Bcl-2 translocation,while germinal center B cells were utilized as the control group.Differential expression analysis,gene co-expression change analysis,GO enrichment analysis,and KEGG pathway enrichment analysis of the mRNA transcriptome data were conducted using the R programming language,with the limma,WGCNA,and clusterProfiler packages.Protein interaction network analysis of differentially expressed genes(DEGs) was performed using STRING database,and the core genes were identified via Cytoscape software,employing CytoHubba and CytoNca plug-ins.The mRNA transcriptomes of IM-9 and DOHH-2 cell lines were sequenced using The Next Generation Sequencing.The core genes were analyzed by paired samplettest based on Read count values,and genes exhibiting statistically significantP-values were identified as the core disease-associated genes.Furthermore,a subset of key genes was selected for validation through Western Blot.Results:A total of 835 DEGs were obtained by differential analysis,and WGCNA obtained a module that was highly correlated with c-MYC/Bcl-2 translocated DLBCL(turquoise module,cor = 0.86,P-value <0.05).GO enrichment analysis showed that a total of 1 437 biological process BP,123 cell component CC and 147 molecular function-related MF were enriched.KEGG enrichment identified 72 related pathways,which were mainly related to ECM receptor interaction,B cell receptor signaling pathway and PI3K-Akt signaling pathway.A total of 284 interacting proteins were obtained from the protein interaction network of STRING database,which were further screened by Cytoscape software.The Next Generation Sequencing and Western Blot verified that COL1A1,COL3A1,COL1A2,MMP2,COL5A1,COL5A2,COL4A2,TIMP1,MMP9,POSTN,BGN,DCN and LUM were the core genes of this type of lymphoma,which affected the survival,invasion and migration of this type of lymphoma cells.Conclusion:To investigate the core genes associated with the pathogenesis,invasion,and migration of c-MYC/Bcl-2 translocated DLBCL cells through the integration of bioinformatics and fundamental experimental methods,with the ultimate goal of laying a theoretical foundation for deeper insights into the mechanisms underlying the initiation and progression of this specific lymphoma subtype,as well as the identification of potential targeted therapeutic agents.