基于生物信息学和转录组测序分析结直肠癌5-FU耐药关键靶点  

Bioinformatics and transcriptome sequencing analysis of 5-FU resistance key targets in colorectal cancer

作　　者：谢竹夫 姚菲 吴清明[1,3] XIE Zhufu;YAO Fei;WU Qingming(Colledge of Medicine,Wuhan University of Science and Technology,Hubei Wuhan 430070,China;Hubei Province Key Laboratory of Occupational Hazard Identification and Control,Hubei Wuhan 430070,China;Tianyou Hospital Affiliated to Wuhan University of Science and Technology,Hubei Wuhan 430061,China)

机构地区：[1]武汉科技大学医学院,湖北武汉430070 [2]职业危害识别与控制湖北省重点实验室,湖北武汉430070 [3]武汉科技大学附属天佑医院,湖北武汉430061

出　　处：《现代肿瘤医学》2022年第23期4226-4232,共7页Journal of Modern Oncology

基　　金：国家自然科学基金面上项目(编号:81573239)。

摘　　要：目的:筛选结直肠癌5-氟尿嘧啶(5-FU)耐药靶基因,并探究该基因对结直肠癌耐药细胞的作用及其作用机制。方法:通过GEO数据库分析GSE28702芯片筛选结直肠癌患者5-FU耐药与5-FU敏感的差异表达基因(differentially expressed genes,DEGs);利用GO与KEGG数据库对DEGs进行通路富集分析;通过STRING数据库构建蛋白质相互作用(protein-protein interaction,PPI)网络,利用Cytoscpae的cytohubba工具筛选枢纽基因。构建5-FU耐药细胞株HCT8/5-FU,利用高通量转录组测序鉴定;采用受试者工作特征曲线(receiver operating characteristic curve,ROC)对诊断价值进行评价并基于TCGA数据库COAD数据集分析关键基因表达及预后;构建TF基因过表达载体转染到HCT8/5-FU细胞系,MTT法检测细胞活力;流式细胞术测定细胞凋亡与细胞周期;qRT-PCR与Western Blot检测基因mRNA和蛋白水平。结果:共筛选出239个DEGs;DEGs主要富集在细胞外囊泡、内吞囊泡腔、药物代谢等通路;PPI得到20个枢纽基因;转录组学显示转铁蛋白(transferrin,TF)在耐药株中显著下调(P<0.05)与生物信息学分析得到的DEGs中TF基因改变趋势相同;与正常结直肠组织相比,癌组织中TF低表达(P<0.05);TF高表达患者总体生存期更长(P<0.05);上调TF表达增强了耐药细胞的5-FU敏感性(P<0.05);上调TF表达增加了5-FU诱导的细胞凋亡与G_(0)/G_(1)期的阻滞(P<0.05);上调TF表达抑制了耐药细胞ABCC1的表达(P<0.05)。结论:基于生物信息学和转录组测序筛选出结直肠癌5-FU耐药靶基因TF,TF基因可能通过调控ABCC1表达降低结直肠癌5-FU的耐药性。Objective:To screen the 5-FU drug resistance target gene in colorectal cancer,and to explore the function of this gene in colorectal cancer drug-resistant cells and its mechanism.Methods:GSE28702 was used to screen the differentially expressed genes(DEGs)of 5-FU-resistant and 5-FU-sensitive colorectal cancer patients by GEO database.GO and KEGG database were used to identify the pathway enrichment analysis on DEGs.PPI network was constructed through STRING database,and hub genes were screened by cytohubba tool of Cytoscpae.5-FU-resistant cell line HCT8/5-FU was constructed,and high-throughput transcriptome sequencing was used to identify differential expression of hub genes.ROC was used to evaluate the diagnostic value,and key gene expression and prognosis were analyzed based on the COAD data set of TCGA database.TF gene overexpression vector was constructed and transfected into HCT8/5-FU cell line,and cell viability was detected by MTT assay.Flow cytometry was used to determine cell apoptosis and cell cycle.qRT-PCR and Western Blot were used to detect mRNA and protein of gene.Results:A total of 239 DEGs were screened.DEGs were mainly enriched in extracellular vesicles,endocytic vesicle lumen,drug metabolism and other functions pathway.20 hub genes were obtained from PPI.Transcriptomics showed that transferrin(TF)was significantly down-regulated in drug-resistant strains(P<0.05),and the change trend of TF gene in DEGs was the same as that obtained by bioinformatics analysis.Compared with normal colorectal,the cancer tissues had low TF expression(P<0.05),and patients with high TF expression had longer overall survival(P<0.05).Up-regulation of TF improved the 5-FU sensitivity of drug-resistant cells(P<0.05).Up-regulation of TF promoted 5-FU-induced apoptosis and G_(0)/G_(1) phase arrest(P<0.05).Up-regulation of TF inhibited the expression of ABCC1 in drug-resistant cells(P<0.05).Conclusion:The 5-FU drug resistance target gene TF in colorectal cancer was screened based on bioinformatics and transcriptome sequencing

关 键 词：5-FU耐药 结直肠癌 生物信息学 转录组测序 转铁蛋白 

分 类 号：R735.3[医药卫生—肿瘤]