基于转录组测序对胆汁酸诱导肝细胞反应的Hub基因的筛选及验证  

Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing

作　　者：张孟妮 张晓珣 欧阳佳凤 柴进 吴晓玲 ZHANG Mengni;ZHANG Xiaoxun;OUYANG Jiafeng;CHAI Jin;WU Xiaoling(College of Medicine,Southwest Jiaotong University,Chengdu,Sichuan Province,610031;Department of Gastroenterology,Cholestatic Liver Diseases Center and Center for Metabolic Associated Fatty Liver Disease,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Gastroenterology,General Hospital of Western Theater Command,Chengdu,Sichuan Province,610083,China)

机构地区：[1]西南交通大学医学院,成都610031 [2]陆军军医大学(第三军医大学)第一附属医院消化内科,胆汁淤积肝病中心和代谢性肝病中心,重庆400038 [3]西部战区总医院消化内科,成都610083

出　　处：《陆军军医大学学报》2023年第18期1883-1893,共11页Journal of Army Medical University

基　　金：国家自然科学基金青年科学基金(82000545);国家自然科学基金面上项目(81770583)。

摘　　要：目的通过转录组测序分析结合型胆汁酸诱导肝细胞的基因表达情况,筛选和验证结合型胆汁酸诱导肝细胞代谢的Hub基因。方法使用牛磺胆酸(taurocholate acid,TCA)处理野生型小鼠的原代肝细胞,转录组测序(RNA sequencing,RNA-Seq)检测经TCA诱导后肝细胞的基因表达情况,将Fold Change≥1.5且P<0.05作为筛选标准获取差异表达基因(differentially expressed genes,DEGs)并进行生物信息学分析。使用STRING(search tool for the retrieval of interesting genes)数据库建立DEGs的蛋白质-蛋白质互作网络(protein-protein interaction,PPI),基于PPI网络利用Cytoscape软件找寻Hub基因。利用实时荧光定量PCR(RT-qPCR)在体内外水平对目标Hub基因进行验证,使用蛋白质免疫印迹试验检测肝细胞内PSAT1/AKT/GSK3β信号通路相关蛋白的表达,使用糖原检测试剂盒检测肝细胞和肝组织糖原水平。结果TCA处理小鼠原代肝细胞共有差异基因293个,其中上调基因122个,下调基因171个。基因本体(Gene Ontology,GO)功能富集分析、京都基因与基因百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析及基因集富集分析(Gene Set Enrichment Analysis,GSEA)的结果显示,DEGs主要富集在代谢、炎症、肿瘤等通路。使用Cytohubba插件得到16个Hub基因,包括Ccnd1、Ppara、Irs1、Ccl2、Ddit3、Serpine1、Nr0b2、Atf3、Phgdh、Trib3、Lepr、G6pc、Psat1、Cxcl2、Asns、Fgf21。RT-qPCR检测结果显示,多种结合型胆汁酸包括TCA、甘氨鹅去氧胆酸(glycochenodeoxycholic acid,GCDCA)、甘氨胆酸(glycocholic acid hydrate,GCA)均能诱导小鼠原代肝细胞内Psat1 mRNA表达水平增高(P<0.05)。此外,胆管结扎(bile duct ligation,BDL)胆汁淤积小鼠肝脏中的Psat1 mRNA水平增高(P<0.05)。Western blot检测结果显示,TCA能够诱导肝细胞内Psat1表达及激活AKT/GSK3β信号通路。TCA能够诱导小鼠原代肝细胞糖原合成增加,与BDL小鼠模型肝组织糖原合成增加以及�Objective To screen and verify the Hub genes involved in conjugated bile acid-induced hepatocyte metabolism through transcriptome sequencing analysis.Methods Primary mouse hepatocytes were treated with taurocholic acid(TCA),and the gene expression of TCA-induced hepatocytes was detected by RNA-Seq analysis.Differential expression genes(DEGs)were identified using a fold change≥1.5 and P value<0.05 as the screening criteria.The protein-protein interaction(PPI)network of DEGs was constructed using the STRING database,and Hub genes were screened using Cytoscape software based on the PPI network.Real-time qPCR was used to validate the identified Hub gene in vitro and in vivo.Western blotting was employed to detect the expression of proteins related to PSAT1/AKT/GSK3βsignaling pathway.The glycogen level in the hepatocytes and liver tissues were detected using glycogen detection kit.Results RNA-Seq analysis identified 293 DEGs in primary mouse hepatocytes upon TCA treatment,including 122 up-regulated genes and 171 down-regulated genes.Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,and Gene Set Enrichment Analysis(GSEA)revealed that these DEGs were mainly enriched in metabolism,inflammatory and tumorigenic pathways.Cytohubba plugin identified 16 Hub genes,including Ccnd1,Ppara,Irs1,Ccl2,Ddit3,Serpine1,Nr0b2,Atf3,Phgdh,Trib3,Lepr,G6pc,Psat1,Cxcl2,Asns,and Fgf21.RT-qPCR results demonstrated that various conjugated bile acids,glycocholic acid(GCA),glycochenodeoxycholic acid(GCDCA),and TCA can induce a significant increase in Psat1 mRNA level in primary mouse hepatocytes(P<0.05).In vivo experiments also showed that the mRNA level of Psat1 was increased significantly in the liver tissues of mouse cholestatic model induced by bile duct ligation(BDL)when compared to the control group(P<0.05).Western blotting indicated that TCA treatment induced the expression of Psat1 and activation of AKT/GSK3βsignaling pathway in primary mouse hepatocytes.TCA treatment also resulted in a

关 键 词：胆汁淤积 糖代谢 小鼠原代肝细胞 转录组测序 

分 类 号：R322.47[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学] R394.3