TLR9通路激活对原代肾小管上皮细胞转录组的影响  

Transcriptome changes upon Toll-like receptor 9 pathway activation in primary renal tubular epithelial cells

作　　者：李一鸣[1] 徐冬雪 张婧[1] 锁进猛 姜军 钱瑶瑶 彭志勇[1] Li Yiming;Xu Dongxue;Zhang Jing;Suo Jinmeng;Jiang Jun;Qian Yaoyao;Peng Zhiyong(Department of Critical Care Medicine,Zhongnan Hospital of Wuhan University,State Key Clinical Specialty of Critical Care Medicine,Hubei Clinical Research Center of Critical Care Medicine,Wuhan 430071,Hubei,China)

机构地区：[1]武汉大学中南医院重症医学科,重症医学国家临床重点专科,湖北省重症医学临床医学研究中心,武汉430071

出　　处：《中华危重病急救医学》2022年第4期394-399,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81971816,81772046,82102273)。

摘　　要：目的探讨Toll样受体9(TLR9)信号通路激活对肾小管细胞转录组的影响。方法提取并培养小鼠原代肾小管上皮细胞,在细胞融合度达80%时分为两组,分别加入10μL磷酸盐缓冲液(PBS,PBS对照组)和终浓度为5μmol/L的TLR9激活剂胞嘧啶-鸟嘌呤寡脱氧核苷酸(CpG-ODN,CpG-ODN处理组)。提取细胞RNA后在Illumina平台进行测序,使用差异基因分析软件DEGseq分析两组细胞中基因的差异表达情况,通过Goatools和KOBAS在线软件分析差异基因所参与的信号通路,应用Homer软件预测转录因子。结果与PBS对照组相比,CpG-ODN处理后有584个显著的差异表达基因,其中102个基因表达上调,482个表达下调。差异表达基因富集最显著的基因本体(GO)为β-干扰素响应、病毒响应或防御等炎症反应相关条目;京都基因与基因组百科全书数据库(KEGG)信号通路富集结果显示,富集系数最显著的信号通路包括2’-5’-寡腺苷酸合成酶活性、核糖核酸酶活性的调节、病毒生命周期的负调控、β-干扰素响应和对原生动物的防御反应等。转录因子预测结果显示,干扰素调节因子3(IRF3)是差异基因启动子序列上富集最显著的转录因子;IRF3是TLR9下游表达差异最显著的转录因子,转录因子21(TCF21)、锌指蛋白135(ZNF135)和阳性调节域4(PRDM4)等转录因子可能是TLR9信号通路的新候选靶标。结论CpG-ODN激活TLR9信号通路,原代肾小管上皮细胞能直接响应CpG-ODN的刺激并发生转录组学变化,为进一步探究TLR9信号通路在脓毒症相关性急性肾损伤中的分子机制研究提供了基础。Objective To explore the effect of Toll-like receptor 9(TLR9)signaling pathway activation on the transcriptome in the renal tubular cells.Methods Mouse primary renal tubular epithelial cells were extracted and cultured.When the degree of cell fusion reached 80%,they were divided into two groups,which were added with 10μL phosphate buffered saline(PBS,PBS control group)and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide(CpG-ODN)with a final concentration of 5μmol/L(CpG-ODN treatment group).The RNA sequencing was performed on the Illumina platform after extraction.DEGseq software was used to analyze the differential expression of genes between the two groups.Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways.Homer software was used to predict transcription factors.Results Compared with the PBS control group,there were a total of 584 differentially expressed genes in the CpG-ODN treatment group,of which 102 were up-regulated and 482 were down-regulated.The most significantly enriched gene ontology(GO)terms of differentially expressed genes included response to interferon-β,defense response to virus and other inflammatory pathway.The most significantly enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathways included 2'-5'-oligoadenylate synthase activity,regulation of ribonuclease activity,negative regulation of virus life cycle,cellular response to interferon-βand defense response to protozoan.The results of transcription factor prediction showed that interferon regulatory factor 3(IRF3)was the most significantly enriched transcription factor in the promoter sequence of differential genes;the most significant transcription factor downstream of TLR9 was IRF3,and other predicted transcription factors such as transcription factor 21(TCF21),zinc finger protein 135(ZNF135),and PR domain containing 4(PRDM4)might be new candidates for TLR9 signaling pathway.Conclusion CpG-ODN activates TLR9 signaling pathway,and primary renal tubular

关 键 词：原代肾小管上皮细胞 胞嘧啶-鸟嘌呤寡脱氧核苷酸 脓毒症相关性急性肾损伤 Toll样受体9通路 

分 类 号：R692[医药卫生—泌尿科学]