Caspase-3抑制剂所致氧化应激状态下肾小管上皮细胞差异表达基因的筛选及功能验证  

Screening and functional verification of differentially expressed genes induced by caspase-3 inhibitor in renal tubular epithelial cells under oxidative stress

作　　者：骆长亮[1] 张秋红 孙林春[3] LUO Changliang;ZHANG Qiuhong;SUN Linchun(Clinical Laboratory,Children’s Hospital of Soochow University,Suzhou 215000;Clinical Laboratory,Suqian Hospital of Nanjing Drum Tower Hospital Group,Suqian 223800;Pediatric Research Center,Children’s Hospital of Nanjing Medical University,Nanjing 210009,China)

机构地区：[1]苏州大学附属儿童医院检验科,江苏苏州215000 [2]南京鼓楼医院集团宿迁医院检验科,江苏宿迁223800 [3]南京医科大学附属儿童医院儿科研究所,江苏南京210008

出　　处：《南京医科大学学报（自然科学版）》2023年第1期17-26,共10页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京市卫生科技发展专项(YKK20125)。

摘　　要：目的:研究caspase-3抑制剂引起的氧化应激状态下肾小管上皮细胞基因表达特点,筛选潜在的候选基因,为揭示caspase-3调控活性氧(reactive oxygen species,ROS)损伤肾小管上皮细胞的机制奠定基础。方法:将人肾小管上皮细胞HK-2用H2O2(300μmol/L)处理6 h后,随机分为对照组和caspase-3抑制剂组(Ac-DEVD-CHO,15μmol/L),运用Illumina Hiseq 2500测序平台完成基因测序,筛选差异基因并进行相关富集分析,用定量PCR对筛选到的前10位差异表达基因(differentially expressed gene,DEG)进行验证。将H2O2(300μmol/L)处理6 h后的HK-2细胞随机分为对照组、caspase-3抑制剂组(Ac-DEVDCHO,15μmol/L)、CTNNB1组[pcDNA3.1(+)-CTNNB1]、caspase-3抑制剂+CTNNB1组[Ac-DEVD-CHO,15μmol/L+pcDNA3.1(+)-CTNNB1]以及caspase-3抑制剂+CTNNB1 NC组[Ac-DEVD-CHO,15μmol/L+pcDNA3.1(+)-CTNNB1 NC],MTT法检测细胞增殖,流式细胞技术检测细胞凋亡和ROS水平,Western blot检测细胞凋亡相关蛋白表达。结果:芯片共筛选出185个DEG,经定量PCR验证,显著性差异排名前10的基因中FIS1、EZR、COL7A1、RPL5、MAP4、CEBPB和CTNNB1 mRNA在caspase-3抑制剂组细胞中低表达(P均<0.05),SNRPB mRNA高表达(P<0.05)。Caspase-3抑制剂组增殖率高于对照组(P<0.05),CTNNB1组增殖率低于对照组(P<0.05),caspase-3抑制剂+CTNNB1组增殖率低于caspase-3抑制剂组(P<0.05),但仍高于对照组(P<0.05)。Caspase-3抑制剂组细胞凋亡率、cleaved-caspase-3和cleaved-PARP蛋白水平低于对照组(P均<0.05),CTNNB1组细胞凋亡率、cleaved-caspase-3和cleaved-PARP蛋白水平高于对照组(P均<0.05),caspase-3抑制剂+CTNNB1组细胞凋亡率、cleaved-caspase-3和cleaved-PARP蛋白水平均较caspase-3抑制剂组高(P<0.05),但仍低于对照组(P<0.05)。caspase-3抑制剂组ROS较对照组降低(P<0.05),CTNNB1组ROS较对照组升高(P<0.05),caspase-3抑制剂+CTNNB1组ROS较caspase-3抑制剂组高(P<0.05),但仍低于对照组(P<0.05)。结论:Caspase-3抑制剂引起的肾小管上�Objective:To study the gene expression characteristics of renal tubular epithelial cells under oxidative stress caused by caspase-3 inhibitors via screening the potential candidate genes,which will lay the foundation for revealing the mechanism of caspase-3regulating ROS injury in renal tubular epithelial cells.Methods:The renal tubular epithelial cells HK-2 were treated with H2O2at a final concentration of 300μmol/L for 6 h,and then randomly divided into control group and caspase-3 inhibitor group(Ac-DEVD-CHO,15μmol/L).The sequencing was completed by using the Illumina Hiseq 2500 platform.Enrichment analysis and an interaction network were performed to screen differential genes.Quantitative PCR was used to verify the first 10 differentially expressed genes(DEGs).The HK-2 cells treated with H2O2(300μmol/L)for 6 h were then divided into control group,caspase-3 inhibitor group(Ac-DEVD-CHO,15μmol/L),CTNNB1 group(pcDNA3.1(+)-CTNNB1),caspase-3 inhibitor+CTNNB1 group(Ac-DEVD-CHO,15μmol/L+pcDNA3.1(+)-CTNNB1)and caspase-3 inhibitor+CTNNB1 NC group(Ac-DEVD-CHO,15μmol/L+pcDNA3.1(+)-CTNNB1 NC)randomly.MTT assay was used to detect cell proliferation,flow cytometry was used to detect cell apoptosis and ROS level,Western blot was used to detect apoptosis-related proteins.Results:A total of 185 DEGs were selected in the control group and the caspase-3 inhibitor group.Quantitative PCR showed FIS1,EZR,COL7A1,RPL5,MAP4,CEBPB and CTNNB1 mRNA were lowly expressed(all P<0.05)and SNRPB mRNA was highly expressed in caspase-3 inhibitor group(P<0.05).The proliferation was higher in the caspase-3 inhibitor group and was lower the CTNNB1 group compared to the control(both P<0.05).The proliferation of the caspase-3 inhibitor+CTNNB1 group was lower than that of the caspase-3 inhibitor group(P<0.05),but still higher than the control(P<0.05).The apoptosis and the expression of cleaved-caspase-3 and cleaved-PARP were lower in caspase-3 inhibitor group and were higher in CTNNB1 group compared to the control(both P<0.05).The apoptosis and t

关 键 词：肾损伤 氧化应激 线粒体 生物信息学 Β-连环蛋白 

分 类 号：R393[医药卫生—基础医学]