miR146a通过Smad4参与多柔比星的心肌细胞毒性作用

miR146a Participates in Doxorubicin Cardiotoxicity Through Smad4

目的:探讨miR146a参与多柔比星心肌细胞毒性作用的可能机制。方法:用多柔比星处理大鼠心肌细胞,用CCK-8方法检测细胞活力,用荧光定量PCR检测miR146a的变化,用Western blot检测切割的Caspase 3的蛋白变化。使用常间回文重复序列丛集(clustered regularly interspaced short palindromic repeats,CRISPR)的方法,设计针对miR146a的向导RNA,敲除miR146a的表达。用CCK-8方法和Western blot分别检测敲除细胞的活力和Caspase 3蛋白变化。用软件预测miR146a的靶基因,利用荧光素酶系统进行验证。用Western blot检测多柔比星处理后靶基因的变化,以及用Western blot检测敲除miR146a对靶基因变化的影响。结果:多柔比星处理导致大鼠心肌细胞活力降低,切割的Caspase 3水平升高,同时发现miR146a表达升高(3.6倍)。使用CRISPR可有效敲除miR146a的表达,敲除效果显著高于microRNA decoy的效果(88.6%vs 57.6%)。敲除miR146a的细胞用多柔比星处理,miR146a增加不明显(1.08倍),并且敲除miR146a抑制了多柔比星导致的细胞活力降低和Caspase 3升高。经生物信息学及荧光素酶检测证实在大鼠心肌细胞内Smad family member 4(Smad4)是miR146a的靶基因,用多柔比星处理心肌细胞后,Smad4的表达降低。而敲除miR146a后,Smad4的表达升高,用多柔比星处理敲除miR146a的细胞,Smad4的表达变化不明显。结论:大鼠心肌细胞中,miR146a通过调节Smad4的表达参与了多柔比星对细胞的毒性作用。

Objective： To investigate the mechanism underlying miR146a participating in the cardiotoxicity of doxorubicin. Methods： Rat cardiomyocytes H9c2 were treated with doxorubicin （DOX）. After that, the cell viability was detected by CCK-8, the miR146a variation was quantified by qPCR and the change of cleaved Caspase 3 was measured by Western blot （WB）. Using CRISPR, two sgRNAs were designed to knockout （KO） the expression of miR146a. The cell viability and cleaved Caspase 3 of miR146a KO cells were respectively detected by CCK-8 and WB. The potential target of miR146a was predicted and validated by luciferase system. The change of target gene after DOX treatment, and the influence of miR146a KO on target gene were detected using WB. Results： After DOX treatment, cell viability decreased, the level of cleaved Caspase 3 elevated and miR146a expression increased 3.6 folds. Using CRISPR, miR146a expression was successfully suppressed, with a much higher inhibitory rate than microRNA decoy （88.6% vs 57.6% ）. miR146a KO significant impeded DOX induced miR146a elevation, cell viability decrement and cleaved Caspase 3 increment. Smad family member 4（Smad4） was proved to be a target of miR146a by informatics prediction and luciferase experiment. The treatment of cardiomyocytes by DOX led to a decrease of Smad4 expression. In miR146a KO cells, the expression of Smad4 increased, which was not changed by DOX treatment. Conclusions： In rat cardiomyocytes, miR146a participated in doxorubicin cardiotoxicity through Smad4.