肾缺血-再灌注损伤circRNA表达的变化及对Hippo信号途径的影响

Differential expression of circRNA in renal ischemia-reperfusion injury and its effect on Hippo signaling pathway

目的分析小鼠肾缺血-再灌注损伤环状RNA(circRNA)及Hippo信号分子YAP的表达变化,初步探讨circRNA对肾缺血-再灌注损伤的作用。方法构建小鼠肾缺血-再灌注损伤模型,检测血清肌酐(Scr)、尿素氮(BUN)及肾组织病理改变判断模型成功。应用Illumina HiSeq 2500系统进行高通量双端测序建立显著差异表达circRNA表达谱。实时定量聚合酶链反应(qRTPCR)验证测序结果及检测相关基因。采用基因功能(GO)分析、通路(KEGG)分析等方法探讨差异表达circRNA参与肾IR损伤的生物功能及作用的主要信号通路。采用蛋白印迹实验检测主要信号分子的表达情况。结果缺血组和对照组间差异显著的circRNA 21个(P<0.05,FC>2),其中10个表达上调,11个表达下调。随机(随机数字法)选取circRNA.1100和cⅡrcRNA.1122进行验证,结果显示肾IR 1d二者的相对表达倍数分别为(0.23±0.016)和(0.36±0.12),相比于对照组均显著降低(P<0.05),与测序数据趋势一致。circRNA.1100在IR 2 d和3 d的肾组织中表达依然显著下调,相对表达倍数分别为(0.24±0.08)和(0.34±0.03)(P<0.05)。circRNA.1100母基因Cpeb3在肾IR 1 d、2 d及3 d的相对表达倍数分别为(0.047±0.022)、(0.098±0.027)和(0.185±0.055),与对照组比较显著下调。生物学功能和通路分析显示差异表达circRNA显著富集在细胞周期、分裂、生长、凋亡、死亡等生物过程及Hippo信号通路。肾IR1d后Hippo通路效应分子YAP蛋白明显上调,持续至IR第3天。结论circRNA参与了肾IR损伤的生理病理过程,差异表达的circRNA及Hippo信号通路可能在肾IR损伤的发生发展中起关键作用。

Objective To analyze the differential expression profile of circRNA and the expression changes of Hippo signaling molecule YAP in renal ischemia-reperfusion injury of mice.Methods A model of renal IR damage in mice was induced,and serum creatinine(Scr)and urea nitrogen(BUN)concentrations and histological changes of samples were detected to assess renal function and tubular injury.Illumina HiSeq 2500 system was used for high-throughput paired-end sequencing to establish the circRNA expression profile with significant differential expression.Real-time quantitative polymerase chain reaction(qRT-PCR)verified the sequencing results and detected related genes.Gene function(GO)and pathway(KEGG)analysis were performed to predict the biological processes and the major signal pathways involved by differentially expressed circRNAs.The expression level of the main signaling molecule was examined by western blot.Results Twenty-one distinctly differentially expressed circRNAs(fold change≥2)were found in IR 24 h kidney tissues compared with the expression in the control groups(P<0.05),among which 10 circRNAs were observed to be up-regulated and 11 down-regulated.CircRNA.1100 and circRNA.1122 were randomly(random number)selected for verification by qRT-PCR,and the relative expressions after renal IR 1day were decreased by(0.23±0.016)and(0.36±0.12),respectively,which were highly consistent with the sequencing trends.Analysis of biological functions and pathways showed that differential expression circRNA was significantly enriched in cell cycles,division,growth,apoptosis,death,and Hippo signaling pathways.The Hippo pathway effector molecule YAP protein was significantly up-regulated after renal IR 1day and until the 3rd day of IR.Conclusions CircRNA may be involved in the regulation of renal IR injury.CircRNA and Hippo pathway may play a key role in the development of renal IR injury.