基于生物信息学的乳腺癌关键基因筛选及实验验证

Bioinformatics screening of breast cancer-related genes and potential drug research

目的:通过利用生物信息学技术,筛选乳腺细胞癌组织与正常组织间的差异表达基因,明确其潜在治疗药物,为将来临床乳腺癌的免疫靶向治疗及药物治疗提供参考。方法:利用基因表达数据库(gene expression omnibus,GEO)检索关键词“乳腺癌”,下载GSE79586芯片数据,通过生信技术筛选对照组及乳腺癌模型组差异表达基因并对其进行基因本体(GO)功能分析、京都基因与基因组百科全书(KEGG)分析、差异基因特征表达分析以及蛋白互作网络(PPI)分析,对分析结果进一步可视化处理。通过GEPIA、GeneMANIA、Timer2.0数据库分别进行预后分析、相关功能预测以及免疫浸润分析。最终通过Connectivity Map(CMap)明确对乳腺癌具有潜在治疗作用的化合物。采用蛋白质印迹法及实时荧光定量PCR(RT-PCR)对核心基因及相关性最高的潜在治疗药物吉非替尼进行体外验证。结果:共筛选到包括1786个上调基因和2130个下调基因在内的3916个差异表达基因;GO结果显示差异基因主要参与磷酸化的正向调节、分泌囊泡、外消旋酶和差向异构酶活性等功能;KEGG结果显示差异基因主要参与系统性红斑狼疮、酒精中毒、粘着斑、阿米巴痢疾等疾病通路与Ras信号通路等;差异基因特征表达分析显示MEK抑制剂、HSP90抑制剂以及信号转导通路激酶抑制剂等是与差异基因相似的药物;PPI结果显示H2AFJ、TFF1、GATA3、FOXA1、CDH1等是与乳腺癌相关的核心基因;进一步选取相关性最高的H2AFJ、TFF1两个核心基因进行GEPIA分析。分析结果显示,原发性乳腺癌细胞组织中H2AFJ与TFF1 mRNA表达显著高于正常组织,并且与原发性乳腺癌细胞患者的病理分期、总生存率以及无病生存率存在显著相关性(P<0.01);H2AFJ与TFF1可能是影响乳腺癌细胞患者生存的潜在预后生物标志物;差异表达的H2AFJ与TFF1功能主要分别与激素受体结合、上皮结构维持和基因表观遗传负调控、参与转录负调控的染色质组织等过程有关;免疫浸润结果显示,H2AFJ与TFF1的表达与巨噬细胞、中性粒细胞、单核细胞、CD4^(+)T、CD8^(+)T以及B淋巴细胞等免疫细胞的浸润存在着显著相关性;CMap结果显示吉非替尼、阿培利西、索拉菲尼、舒尼替尼等化合物对乳腺癌具有潜在的治疗作用。Western blot和RT-PCR结果显示,H2AFJ与TFF1在乳腺癌细胞中显著高表达,吉非替尼可明显抑制乳腺癌细胞中H2AFJ与TFF1的表达(P<0.05,P<0.01)。结论:本研究通过生物信息学手段筛选出乳腺细胞癌组织与正常组织间差异表达基因,明确在乳腺癌发病进程中的关键基因及具有潜在治疗效果的化合物,进一步通过实验验证了所筛选药物对乳腺癌的有效性。为今后临床针对乳腺癌的新药研发提供参考,以便开发更有效的治疗方案。

Objective:To search the the differentially expressed genes between breast cell carcinoma tissues and normal tissues by using bioinformatics technology,and the potential therapeutic drugs for breast cancer were identified,which can provide reference for clinical immune targeted therapy and drug therapy of breast cancer in the future.Methods:"Breast cancer"was searched by using Gene Expression Omnibus(GEO),and GSE79586 chip data was downloaded.The differentially expressed genes in the control group and the breast cancer model group were screened by using bio-communication technology and subjected to GO function analysis,KEGG pathway analysis,differential gene characteristic expression analysis and protein-protein interaction network(PPI)analysis,and the analysis results were further visualized.Prognosis analysis,related function prediction and immune infiltration analysis were performed using the GEPIA,GeneMANIA,and Timer2.0 databases,respectively.Finally,the compounds with potential therapeutic effects on breast cancer are identified through Connectivity Map(CMap).Western blotting and real-time PCR(RT-PCR)were used to verify the core genes and potential therapeutic agents with the highest correlation in vitro.Results:A total of 3916 differentially expressed genes including 1786 up-regulated genes and 2130 down-regulated genes were screened.GO analysis showed that the differential genes were mainly involved in the positive regulation of phosphorylation,secretory vesicles,racemase and epimerase activities.KEGG analysis showed that differential genes were involved in systemic lupus erythematosus,alcoholism,sticky spots,amoebic dysentery Ras signal pathways and other disease pathways.The characteristic expression analysis of differential genes showed that MEK inhibitors,HSP90 inhibitors and signal transduction pathway kinase inhibitors were drugs similar to the differential genes.PPI results showed that H2AFJ,TFF1,GATA3,FOXA1,and CDH1were core genes related to breast cancer.Two core genes of H2AFJ and TFF1 with the highest correlation were further selected for GEPIA analysis.The results of the analysis showed that the mRNA expression levels of H2AFJ and TFF1 in breast cancer cells were significantly higher than those in normal tissues,and there was a significant correlation with the pathological staging,overall survival rate and disease-free survival rate of breast cancer patients.H2AFJ and TFF1 may be potential prognostic biomarkers for survival of breast cancer patients.The functions of differentially expressed H2AFJ and TFF1 are mainly related to hormone receptor binding,epithelial structure maintenance and epigenetic negative regulation of genes,chromatin tissue involved in negative regulation of transcription,etc.The results of immune infiltration showed that the expressions of H2AFJ and TFF1 had a significant correlation with the infiltration of macrophages,neutrophils,monocytes,CD4^(+)T,CD8^(+)T,B lymphocytes and other immune cells.CMap results showed that compounds such as Gefitinib,Alpelisib,Sorafenib,and Sunitinib had potential therapeutic effects on breast cancer.Western blot and RT-PCR results showed that H2AFJ and TFF1 were significantly overexpressed in breast cancer cells.Gefitinib significantly inhibited the expression of H2AFJ and TFF1 in breast cancer cells(P<0.05,P<0.01).Conclusion:In this study,differentially expressed genes between breast cell carcinoma tissues and normal tissues were screened out by bioinformatics means to further identify key genes and compounds with potential therapeutic effects in the onset process of breast cancer and to further verify the effectiveness of the screened drugs on breast cancer through experiments.It will provide reference for clinical research and development of new drugs against breast cancer in the future in order to develop more effective treatment options.