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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 possible mechanism of miR146a involved in the cytotoxicity of doxorubicin-induced myocardial cells. Methods: Cardiomyocytes were treated with doxorubicin, cell viability was detected by CCK-8, the changes of miR146a were detected by fluorescence quantitative PCR, and the changes of cleaved Caspase 3 protein were detected by Western blot. MiR146a was knocked down and the guide RNAi targeting miR146a was designed and synthesized using the clustering regularly interspaced short palindromic repeats (CRISPR). The activity of knock-out cells and the changes of Caspase 3 protein were detected by CCK-8 and Western blot respectively. The target gene of miR146a was predicted by software and verified by the luciferase system. Western blot was used to detect the change of target gene after doxorubicin treatment, and Western blot was used to detect the effect of miR146a knockdown on target genes. RESULTS: Doxorubicin treatment resulted in decreased cardiomyocyte viability, increased cleaved Caspase 3 levels, and increased miR146a expression (3.6 fold). The knockdown effect of miR146a knockdown was significantly higher than that of microRNA decoy (88.6% vs 57.6%) using CRISPR. MiR146a knockdown cells treated with doxorubicin showed no significant increase in miR146a (1.08 fold), and knockdown of miR146a inhibited doxorubicin-induced decrease in cell viability and Caspase 3 elevation. Bioinformatics and luciferase assay confirmed that Smad family member 4 (Smad4) was the target gene of miR146a in rat cardiomyocytes. The expression of Smad4 was decreased after treatment of cardiomyocytes with doxorubicin. After miR146a knockdown, the expression of Smad4 was up-regulated. The knockdown of miR146a cells with doxorubicin showed no significant change in Smad4 expression. Conclusion: miR146a is involved in the cytotoxicity of doxorubicin in rat cardiomyocytes by regulating Smad4 expression.