miR-125b-5p通过靶向钙通道电压依赖性β1蛋白调控心衰模型心肌细胞凋亡的机制研究

miR-125b-5p regulates cardiomyocyte apoptosis in heart failure model by targeting CACNB1: a mechanistic study

目的探讨miR-125b-5p对心衰心肌细胞凋亡的影响及其潜在机制。方法分离培养大鼠原代心肌细胞,采用0.8%戊巴比妥钠诱导心肌细胞构建心衰模型。实时荧光定量PCR(qRT-PCR)和免疫印迹实验检测心衰心肌细胞中miR-125b-5p和钙通道电压依赖性β1蛋白(CACNB1)表达。下调miR-125b-5p或过表达CACNB1,流式细胞仪检测细胞凋亡。Targetscan在线预测、双荧光素酶报告基因实验和免疫印迹验证miR-125b-5p和CACNB1的靶向关系。将miR-125b-5p inhibitors和CACNB1 siRNA共转染至心衰心肌细胞,流式细胞仪检测细胞凋亡。结果与对照组相比,心衰心肌细胞中miR-125b-5p表达上调(3.11±0.12比1.04±0.09,P<0.05),而CACNB1的mRNA和蛋白表达显著下调(0.76±0.07比1.96±0.11、0.53±0.05比0.94±0.06,均P<0.05)。下调miR-125b-5p或过表达CACNB1,心衰心肌细胞凋亡率显著降低[(10.11±0.92)%比(19.76±1.07)%、(8.11±0.67)%比(21.76±1.22)%,均P<0.05]。Targetscan在线预测、双荧光素酶报告基因实验和免疫印迹实验结果表明,miR-125b-5p可靶向调控CACNB1蛋白表达。敲减CACNB1可逆转miR-125b-5p对心衰心肌细胞凋亡的抑制作用。结论miR-125b-5p可通过靶向调节CACNB1表达,抑制心衰大鼠心肌细胞凋亡。

Objective To investigate the effect of miR-125b-5p on the apoptosis of cardiomyocytes in heart failure and its potential mechanism. Methods Rat primary cardiomyocytes were isolated and cultured to establish heart failure model induced by 0.8% pentobarbital sodium. Real-time quantitative PCR (qRT-PCR) and Western blotting were used to determine the protein expression of miR-125b-5p and voltage-dependent calcium channel β1 (CACNB1) in cardiomyocytes of heart failure model, respectively. After down-regulation of miR-125b-5p or overexpression of CACNB1, the apoptosis was examined by flow cytometry. The targeting relationship between miR-125b-5p and CACNB1 was verified by Targetscan online prediction, dual luciferase reporter gene assay and Western blotting. The miR-125b-5p inhibitors and CACNB1 siRNA were co-transfected into heart failure cardiomyocytes, and the apoptosis was determined by flow cytometry. Results Compared with the control group, the expression level of miR-125b-5p was up-regulated in heart failure cardiomyocytes (3.11±0.12 vs. 1.04±0.09, P<0.05), while the mRNA and protein expression levels of CACNB1 were significantly down-regulated (0.76±0.07 vs. 1.96±0.11, 0.53±0.05 vs. 0.94±0.06, both P<0.05). After down-regulation of miR-125b-5p or overexpression of CACNB1, the apoptosis rate of heart failure cardiomyocytes decreased significantly[(10.11±0.92)% vs.(19.76±1.07)%,(8.11±0.67)% vs.(21.76±1.22)%, both P<0.05]. The findings of Targetscan online prediction, dual luciferase reporter gene assay and Western blotting indicted that miR-125b-5p could target-regulate the protein expression of CACNB1. Knockdown of CACNB1 could reverse the inhibition of miR-125b-5p on cardiomyocyte apoptosis in heart failure. Conclusion miR-125b-5p may inhibit the apoptosis of cardiomyocytes in rats with heart failure by target-regulation of CACNB1 expression.