微小RNA-130b在嘌呤霉素氨基核苷诱导的足细胞损伤中的作用及机制

Role and its mechanism of micro RNA-130b in puromycin aminonucleoside-induced podocyte injury

目的探讨微小RNA-130b(miR-130b)在嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)诱导的足细胞损伤中的作用及机制。方法小鼠肾脏足细胞系MPC5予以PAN 25、50、100μg/mL干预,实时定量PCR检测MPC5细胞系内miR-130b及其宿主基因2610318N02Rik(RIK)表达;转染miR-130b抑制剂(miR-130b inhibitor)或对照(NC inhibitor)后予以PAN 100μg/mL刺激,24 h后采用Western blot检测足细胞裂孔膜蛋白Synaptopodin和Nephrin蛋白表达变化;采用鬼笔环肽染色检测足细胞骨架改变;采用流式细胞技术检测细胞凋亡变化;采用Targetscan 7.2预测miR-130b潜在靶基因,Western blot检测miR-130b模拟物(mimic)对于潜在靶基因PGC1α蛋白表达的影响;采用双荧光素酶报告基因系统检测miR-130b mimic对于荧光活性的影响。结果PAN可以抑制足细胞内的miR-130b及宿主基因RIK表达;与转染NC inhibitor组比较,miR-130b inhibitor提高足细胞裂孔膜蛋白synaptopodin和nephrin表达;鬼笔环肽染色显示抑制miR-130b可以部分缓解PAN诱导的足细胞骨架紊乱及重组;流式细胞技术检测结果示干扰足细胞miR-130b表达可以减少PAN诱导的细胞凋亡;Targetcan 7.2分析显示PGC1α是miR-130b的潜在靶基因,Western blot结果显示,转染miR-130b mimic可以降低足细胞内PGC1α表达;双荧光素酶报告基因系统结果示,与NC mimic及突变的PGC1α3’-UTR共转染组比较,miR-130b mimic与野生型PGC1α3’-UTR共转染组荧光素酶活性降低。结论miR-130b通过靶向抑制PGC1α表达参与PAN诱导的足细胞损伤。

Objective To investigate the role and its mechanism of miR-130b in podocyte injury induced by puromycin aminonucleoside(PAN).Methods The immortalized mouse podocytes MPC5 were treated by 25,50,100μg/mL PAN,then real-time quantitative polymerase chain reaction(PCR)was used to detect the expression of miR-130b and the host gene 2610318N02Rik(RIK);MPC5 were transfected with MiR-130b inhibitor or normal control(NC)inhibitor,then the cells were stimulated with 100μg/mL PAN for 24 h,then western blot was used to detected the protein expression of Synaptopodin and Nephrin.Phalloidin dying was used to detect the changes of cytoskeleton.Flow cytometry was used to measure podocyte apoptosis.Targetscan 7.2 was adopted to predict the potential target gene of miR-130b,and Western blot was used to detect effect of miR-130b mimic on expression of the potential target gene PGC1a.Dual-luciferase reporter assay system was utilized to detect effect of miR-130b mimic on fluorescence activity.Results PAN significantly inhibited the expression of miR-130b and RIK.The western blot showed that inhibition of miR-130b increased the protein expression of Synaptopodin and Nephrin compared to NC inhibitor group.Phalloidin dying showed that inhibition of miR-130b partially alleviated cytoskeletal remodeling of podocytes induced by PAN.Flow-cytometric analysis showed that PAN-induced apoptosis was decreased after miR-130b silencing;Targetcan 7.2 analysis showed miR-130b mimic could significantly down-regulate the protein expression of PGC1α,the dual luciferase reporter assay system results showed that miR-130b and mild PGC1α3’-UTR co-transfection decreased PGC1α3’-UTR luciferase activity compared to the control mimic group and the mutant PGC1α3’-UTR co-transfection group,but there was no significant difference between the control mimic group and the mutant·PGC1α3’-UTR group.Conclusions MiR-130b involves in podocyte injury induced by PAN by target inhibition of PGC1αexpression.