摘要
目的:获得马兜铃酸I(Aristolochic acid I,AAI)致肾小管上皮细胞损伤的动态差异转录组,为进一步探讨AAI肾毒性分子机制奠定基础。方法:基于CCK8和检测LDH活性,采用136.51μg/mL AAI孵育HK-2细胞0、3、6、12、24 h,提取细胞样本总RNA并进行高通量转录组测序,计算基因在不同样本中的表达量;筛选不同时间点与0 h间差异基因,筛选条件为差异倍数大于2且P值小于0.05;应用基因本体论(gene ontolog,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)数据库进行基因的功能分析;通过韦恩图分析不同时间点的共有和特有差异基因,利用qRT-PCR验证随机挑选的差异基因的表达量,验证是否与转录组测序结果的变化趋势一致。结果:全部样本共获得115.48 G的干净序列。与对照组相比,3、6、12、24 h不同时间点差异基因数量分别为1535、2406、3439、5073个,其中上调基因分别为920、1405、1675、2623个,下调基因分别为615、1001、1764、2450个。差异基因中共有2873个获得GO数据库功能注释,主要涉及生物过程、细胞组成与分子功能3个大类。KEGG分析发现上调差异基因富集的条目主要是神经活性配体-受体相互作用、苯丙氨酸代谢、癌症的转录失调等,下调差异基因富集的条目主要涉及细胞因子-细胞因子受体相互作用、TNF信号通路、Wnt信号通路等。结论:本研究得到大量时效转录组数据库和差异表达基因,对不同时间点共有差异基因进行了功能注释与PCR验证,表明马兜铃酸I的肾小管上皮细胞毒性过程涉及神经活性配体-受体相互作用、苯丙氨酸代谢、TNF信号通路等环节。
Objective:To reveal the dynamic differential transcriptomes in aristolochic acid I(AAI)-induced injury of renal tubular epithelial cells(RTECs),so as to lay a foundation for exploring the molecular mechanism of AAI-induced nephrotoxicity.Methods:Based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and lactate dehydrogenase(LDH)release assays,HK-2 cells were incubated with 136.51μg/mL AAI for 0,3,6,12,and 24 h,respectively.The total RNA of cells was extracted and subjected to high-throughput transcriptome sequencing,followed by the calculation of gene expression in different samples.The differential genes between different time points and 0 h were screened out under the screening condition of fold change greater than 2 and P value less than 0.05.The gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway analyses were performed to disclose functional enrichment of genes.Venn diagram was used to analyze the common and unique differential genes at different time points.qRT-PCR was conducted to validate the transcriptome results based on randomly selected differential genes.Results:A total of 115.48 G of clean reads were obtained.Compared with the control,the number of differential genes at 3,6,12 and 24 h was 1535,2406,3439 and 5073,respectively,among which 920,1405,1675 and 2623 genes were up-regulated,while 615,1001,1764 and 2450 were down-regulated,respectively.A total of 2873 genes were annotated with GO terms,which were classified into biological process,cell composition and molecular function.KEGG analysis showed that the up-regulated differential genes were mainly enriched in neuroactive ligand-receptor interaction,phenylalanine metabolism,and transcriptional disorders in cancer,while the down-regulated differential genes mainly in cytokine-cytokine receptor interaction,TNF signaling pathway,and Wnt signaling pathway.Conclusion:In this study,a large number of aging transcriptome databases and differential genes were obtained.The functional annotation of differential genes at different time points and PCR validation have confirmed that AAI produces cytotoxicity in RTECs via neuroactive ligand-receptor interaction,phenylalanine metabolism,TNF signaling pathway,etc.
作者
王静
黄恺
朱哿瑞
马园园
刘成海
陶艳艳
Wang Jing;Huang Kai;Zhu Gerui;Ma Yuanyuan;Liu Chenghai;Tao Yanyan(Institute of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203;Shanghai Key Laboratory of Traditional Chinese Medicine,Shanghai 201203)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2021年第3期41-46,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
“重大新药创制”科技重大专项(编号:2019ZX09201001)
上海市科委科技支撑项目(编号:19401901500)
上海市中医药三年行动计划[编号:ZY-(2018-2020)-CCCX-5001]。
关键词
马兜铃酸
肾小管上皮细胞
转录组
作用机制
细胞毒性
aristolochic acid I(AAI)
renal tubular epithelial cells(RTECs)
transcriptome
mechanism
cytotoxicity