miR-30e*在顺铂诱导的肾近端小管上皮细胞凋亡和线粒体损伤中的作用
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国家自然科学基金资助(81270797)


Role of miR-30e* in cisplatin induced renal proximal tubular cells apoptosis and mitochondrial injury
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    目的:观察顺铂诱导肾近端小管细胞损伤过程中miR-30e*的表达变化,并探讨其在顺铂诱导的小管细胞凋亡和线粒体损伤中的作用。方法:实时定量PCR(qRT-PCR)检测顺铂处理后的小管细胞miR-30e*的表达水平;使用重组慢病毒载体感染细胞,稳定高表达或低表达miR-30e*;使用流式细胞仪检测细胞凋亡,观察miR-30e*在顺铂诱导的小管细胞凋亡中的作用;qRT-PCR检测线粒体DNA(mtDNA)拷贝数,JC-1检测线粒体膜电位变化,探究miR-30e*在顺铂引起的线粒体损伤中的作用。结果:①顺铂处理肾小管上皮细胞后,miR-30e*的表达呈时间依赖性和剂量依赖性下调;②使用qRT-PCR检测重组慢病毒载体制备的稳定高表达或低表达miR-30e*的小管细胞株中miR-30e*的表达,过表达组增高了7倍,敲低表达组降低了近50%;③加入顺铂后,与对照组相比小管细胞凋亡增加,过表达miR-30e*对凋亡起保护作用,敲低miR-30e*则加重凋亡;④通过检测mtDNA的拷贝数发现顺铂诱导的线粒体损伤在48 h时有意义,迟于miR-30e*表达的下调,线粒体膜电位检测JC-1提示过表达miR-30e*对线粒体有保护作用。结论:顺铂处理小管细胞可降低其miR-30e*的表达;体外过表达miR-30e*对顺铂引起的小管细胞的凋亡和线粒体损伤具有保护作用,敲低miR-30e*会加重顺铂诱导的肾近端小管细胞凋亡和线粒体损伤。

    Abstract:

    Objective:To investigate the expression of miR-30e* and the role of miR-30e* in cisplatin induced mouse proximal tubular cells (mPTCs) apoptosis and mitochondrial injury. Methods:Expression of miR-30e* in mPTCs treated with cisplatin were determined by quantitative real-time PCR (qRT-PCR). To stably over expression or knock down miR-30e*, recombinant lentiviral expressing vectors were used for transfection of mPTCs. Flow cytometry was performed to analyze the apoptosis of mPTCs after transfection and treated with or without cisplatin. Mitochondrial DNA (mtDNA) copy numbers were determined by qRT-PCR and mitochondrial membrane potential was examined by JC-1 staining. Results:Cisplatin reduced the expression levels of miR-30e* in a dose and time-dependent manner. MPTCs transfected with over expression miR-30e* recombinant lentiviral expressing vector gained an increased expression of miR-30e* for seven fold. While knock down of miR-30e* decreased the expression of miR-30e* for 50% compared with vehicle. The apoptosis of mPTCs increased when treated with cisplatin,over expression miR-30e* reduced cisplatin induced apoptosis and knock down of miR-30e* facilitated cisplatin induced apoptosis. mtDNA significantly decreased after cisplatin treatment for 48 h,later than the decrease expression of miR-30e*. JC-1 staining revealed ectopic miR-30e* can protect mitochondrial membrane from cisplatin induced injury. Conclusion:Cisplatin reduced the expression levels of miR-30e* in vitro. Ectopic miR-30e* can protect mPTCs from apoptosis and mitochondrial membrane potential changes induced by cisplatin.

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杨玲云,郭 燕,倪佳佳,王 荣,林加娟,丁桂霞,黄松明,张爱华. miR-30e*在顺铂诱导的肾近端小管上皮细胞凋亡和线粒体损伤中的作用[J].南京医科大学学报(自然科学版),2015,(8):1060-1065

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  • 收稿日期:2015-01-17
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  • 在线发布日期: 2015-08-04
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