皮肤黑色素瘤差异表达基因的生物信息学分析及调控中药预测

Bioinformatics analysis of differentially expressed genes in cutaneous melanoma and prediction of traditional Chinese medicine regulation

目的 通过生物信息学技术分析皮肤黑色素瘤患者皮肤组织与非癌皮肤组织的基因表达微阵列数据,筛选出皮肤黑色素瘤的差异表达基因,预测可用于诊断和治疗皮肤黑色素瘤的生物标志物及潜在中药。方法 从基因表达数据库(Gene Expression Omnibus,GEO)提取GSE3189、GSE114445、GSE46517基因芯片,使用GEO2R工具筛选差异表达基因(differentially expressed genes,DEGs)。利用DAVID数据库对DEGs进行基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析;使用String数据库构蛋白-蛋白互作网络,应用Cytoscape软件及其插件筛选PPI网络中关键基因,应用GSCA数据库进行关键基因的突变分析。通过Gepia 2.0数据库结合癌症基因组图谱数据集(Cancer Genome Atlas,TCGA)对关键基因进行生存分析。根据度(degree)值和生存分析结果综合筛选出核心基因(集),应用GSCA数据库对核心基因(集)进行泛癌分析和免疫浸润分析,并使用TIMER 2.0数据库对核心基因分别进行免疫浸润分析。使用Coremine Medical数据库预测作用于关键基因的潜在中药。结果 共筛选出DEGs 114个,其中上调基因36个,下调基因78个。上调DEGs主要富集在细胞因子-细胞因子受体相互作用、Toll样受体信号等通路,下调DEGs主要富集在细胞黏附、细胞增殖的正调控等方面。15个关键基因为EGFR、KPNA2、GATA3、AURKA、TYMS、PKP1、SPP1、MMP1、KLF4、EPCAM、KIF23、DTL、CLDN1、LCP2、FGFR2,其中共3个基因KPNA2、AURKA、DTL被视为核心基因(集)。潜在治疗中药有丹参、人参、构皮、高良姜、甘草、蓖麻子、油桐叶等。结论 皮肤黑色素瘤中关键基因的突变以FGFR2的错义突变为主,突变方式多为胞嘧啶(C)向胸腺嘧啶(T)的转换。EGFR、KPNA2、GATA3、AURKA、PKP1、DTL、LCP2对皮肤黑色素瘤的生存预后存在显著影响。核心基因集在多种癌症中显著上调,并与自然调节性T细胞(natural regulatory T-cells,nTreg)细胞、树突状细胞、单核细胞等免疫细胞的浸润有关。潜在调控中药包括补虚药、清热解毒药、拔毒生肌药、消肿拔毒药、温里药、健脾利湿药、利水渗湿药、活血化瘀药、安神药9类,在相关古籍中存在对应的中医内外治经验记载,其中药活性成分亦被证明对皮肤黑色素瘤存在一定的调控和抑制作用。

Objective To screen and analyze the differentially expressed genes(DEGs) between cutaneous melanoma tissues and nontumor tissues with bioinformatics techniques and to predict biomarkers and traditional Chinese medicines(TCMs) for diagnosing and treating cutaneous melanoma.Methods The microarray data sets GSE3189,GSE114445 and GSE46517 were downloaded from the Gene Expression Omnibus(GEO),and GEO2R was used to screen the DEGs.Kyoto encyclopedia of genes and genomes(KEGG)analysis and gene ontology(GO) analysis were performed on DEGs based on the DAVID database.The protein-protein interaction network was constructed by String,Cytoscape and related plug-ins were used to screen the hub genes in the network.GSCA,Gepia 2.0 and Cancer Genome Atlas(TCGA) datasets were used for mutation analysis and survival analysis of hub genes.Core genes(set)were comprehensively screened according to degree value and survival analysis results.The GSCA and TIMER 2.0 databases were used for pan-cancer and immune infiltration analyses of core genes(set).Coremine Medical was applied to predict the TCMs acting on hub genes.Results A total of 114 DEGs were screened out,with 36 up-regulated and 78 down-regulated.The up-regulated genes were mainly involved in cytokine-cytokine receptor interaction and toll-like receptor signaling,while down-regulation genes were mainly concentrated in cell adhesion and positive regulation of cell proliferation.The 15 hub genes were EGFR,KPNA2,GATA3,AURKA,TYMS,PKP1,SPP1,MMP1,KLF4,EPCAM,KIF23,DTL,CLDN1,LCP2,FGFR2,among which KPNA2,AURKA and DTL were regarded as core genes(set).The potentially effective TCMs against cutaneous melanoma were Danshen(Salviae Miltiorrhizae Radix et Rhizoma),Renshen(Ginseng Radix et Rhizoma),Goupi(bark of Broussonetia papeyrifera),Gaoliangjiang(Alpiniae Officinarum Rhizoma),Gancao(Glycyrrhizae Radix et Rhizoma),Bimazi(Ricini Semen),Youtongye(leaves of Vernicia fordii) and so on.Conclusion Mutations of hub genes in cutaneous melanoma were mainly missense mutations of FGFR2,which were mostly caused by cytosine(C) to thymine(T) conversion.EGFR,KPNA2,GATA3,AURKA,PKP1,DTL,and LCP2 have significant effects on the survival prognosis of cutaneous melanoma.The core gene set was significantly up-regulated in various cancers and associated with infiltrating immune cells such as natural regulatory T-cells(nTreg),dendritic cells,and monocytes.The potential effective TCMs can be divided into nine categories,including tonic medicines,heat-clearing and toxin-removing medicines,detoxification and myogenic medicines,discutient medicines,interior-warming medicines,spleen-invigorating medicines,urination-promoting and dampness-draining medicines,blood-circulating and blood stasis-resolving medicines,mind-calming medicines.There are corresponding records of TCMs internal and external treatment experience in relevant ancient books,and the active ingredients of TCMs also prove that they have certain regulation and inhibition on cutaneous melanoma in the pharmacological mechanism.