SIRT1在糖尿病肾病中的诊断及治疗价值

Diagnostic and therapeutic value of SIRT1 in diabetic kidney disease

目的鉴定糖尿病肾病(DKD)新的诊断标志物及治疗靶点。方法基于数据库进行挖掘以及机器学习策略鉴定出新的DKD诊断标志物。通过生物信息学方法预测调控该靶点的上游微小RNA(miRNA),利用双荧光素酶实验验证其与靶点的结合。以BTBR ob/ob小鼠作为2型DKD模型,将小鼠按每组10只分为正常对照组、DKD空白对照组、DKD转染非同源miRNA对照组、DKD转染目标miRNA agomir组,DKD转染目标miRNA antagomir组,探究调控该miRNA和该靶点对DKD肾脏损害、肾脏炎症反应、纤维化及氧化应激的影响。结果共鉴定出100个差异表达基因。通过3种机器学习策略,最终SIRT1被鉴定为DKD的潜在诊断标志物。利用RNAhybird数据库预测发现SIRT1 mRNA 3’-UTR中的碱基序为miR-138-5p提供了结合位点,且双荧光素酶实验确定了miR-138-5p可负向调控SIRT1的表达。动物实验结果发现,miR-138-5p高表达可加重2型DKD小鼠的多尿、高血糖、高脂血症、高尿素氮以及蛋白尿症状,相应的miR-138-5p低表达则减轻了2型DKD小鼠的上述症状。进一步机制研究发现,相较于正常对照组,PAI-1、VCAM-1、TGF-β1、CTGF、ROS以及MDA在2型DKD小鼠肾组织中均表达升高(P<0.05),相较于2型DKD小鼠,这些指标在miR-138-5p高表达组中均升高(P<0.05),在miR-138-5p低表达组中均降低(P<0.05)。结论SIRT1是DKD的潜在诊断及治疗靶点,调控miR-138-5p/SIRT1途径可减轻DKD肾脏损害、肾脏炎症反应、纤维化及氧化应激。

Objective To identify new diagnostic markers and therapeutic targets for diabetic kidney disease(DKD).Methods New DKD diagnostic markers were identified based on database mining as well as machine learning strategies.The upstream microRNAs(miRNAs)regulating the target were predicted by bioinformatics methods,and their binding to the target was verified using dual luciferase assay.BTBR ob/ob mice were used as a model of type 2 DKD.To explore the effects of regulating the miRNA and the target on the renal damage,renal inflammatory response,fibrosis,and oxidative stress,the mice were divided into normal control group,DKD blank control group,DKD transfected non-homologous miRNA control group,DKD transfected target miRNA agomir group,and DKD transfected target miRNA antagomir group by 10 mice in each group.Results A total of 100 differentially expressed genes were identified.Through three machine learning strategies,SIRT1 was finally identified as the potential diagnostic marker for DKD.Using RNAhybird database for prediction,it was found that the base sequence in the 3'-UTR of SIRT1 mRNA provided a binding site for miR-138-5p,and the dual luciferase assay determined that miR-138-5p could negatively regulate the expression of SIRT1.The results of animal experiments revealed that high miR-138-5p expression aggravated the symptoms of polyuria,hyperglycemia,hyperlipidemia,high-blood urea nitrogen,and proteinuria in type 2 DKD mice,while corresponding low miR-138-5p expression alleviated the above symptoms in type 2 DKD mice.Further mechanism studies revealed that PAI-1,VCAM-1,TGF-β1,CTGF,ROS,and MDA were all expressed at elevated levels in renal tissues of type 2 DKD mice compared to normal controls(P<0.05),and that these metrics were all elevated in the miR-138-5p high-expression group(P<0.05)and reduced in the miR-138-5p low expression group(P<0.05).Conclusion SIRT1 is a potential diagnostic and therapeutic target for DKD,and modulation of the miR-138-5p/SIRT1 pathway attenuates renal damage,renal inflammatory response,fibrosis and oxidative stress in DKD.