miR-1、miR-16和miR-208前体在乳鼠心肌细胞和心肌成纤维细胞中的表达

The expression of microRNA-1, -16 and -208 precursor in neonatal mouse and rat cardiomyocytes and cardiac fibroblasts

目的检测心肌特异的miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中的表达。方法利用出生1～3d的Sprague-Dawley(SD)大鼠和C57BL/6小鼠,采用2.5%胰酶消化和差速贴壁法分离心肌细胞和心肌成纤维细胞。提取原代的乳小鼠、乳大鼠心肌细胞及P1代的心肌成纤维细胞总RNA,以β-actin为内参照,逆转录酶-聚合酶链式反应(RT-PCR)分别检测miR-1、miR-16和miR-208的前体表达水平。用FluorChem8900软件分析琼脂糖凝胶电泳后PCR产物条带的灰度值,计算出表达的目的miRNA前体与β-actin的比值。结果有效分离并培养了乳C57BL/6小鼠、乳SD大鼠心肌细胞及心肌成纤维细胞。RT-PCR结果显示,在乳SD大鼠心肌细胞及心肌成纤维细胞中miR-1和miR-16均有相近水平的表达;心肌细胞中miR-208的表达显著低于miR-208b(P<0.01),miR-208b在心肌细胞中的表达显著高于其在心肌成纤维细胞中的表达(P<0.05)。乳C57BL/6小鼠心肌细胞中miR-1a-1的表达显著低于miR-1a-2(P<0.01),而miR-1a-2在心肌细胞和心肌成纤维细胞中的表达水平一致;miR-16-1在心肌细胞和心肌成纤维细胞中表达水平相近,而miR-16-2在两种细胞中均不表达;心肌细胞中miR-208a和miR-208b表达水平差别不明显,心肌成纤维细胞中只有miR-208a表达,并且在水平上低于其在心肌细胞中的表达(P<0.05)。结论 miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中呈细胞特异性的表达。

Objective To detect the myocardium-specific miR-1, miR-16 and miR-208 precursor expressions in neonatal mouse and rat cardiomyocytes and cardiac fibroblasts. Methods Primary cardiomyocytes and cardiac fibroblasts were isolated from 1 to 3-day-old neonatal Sprague-Dawley （SD） rats and C57BL / 6 mice by trypsinization in 2.5% trypsin and a differential adhesion method. Total RNA was extracted from primary cardiomyocytes and the first passage of cardiac fibroblasts by using TRIzol reagent. The expression of miR-1, miR-16 and miR-208 precursor was determined by reverse transcription PCR （RT- PCR）. The intensity of PCR product band was measured by using FluorChem8900 software, and the relative expression level of some miRNA precursor was indicated by the ratio between the miRNA precursor of interest and β-actin gene. Results The primary cardiomyocytes and cardiac fibroblasts were successfully isolated and cultured in our study. The results of RT-PCR showed that miR-1 and miR-16 precursor was expressed in neonatal SD rat cardiomyocytes and cardiac fibroblasts at similar levels, respectively. The precursor level of miR-208 was significantly lower than that of miR-208b in neonatal SD rat cardiomyocytes （P0.01）, and miR-208b precursor expression in neonatal SD rat cardiomyocytes was higher than that in neonatal SD rat cardiac fibroblasts （P0.05）. The precursor level of miR-1a-1 was significantly lower than that of miR-1a-2 in neonatal C57BL / 6 mouse cardiomyocytes （P0.01）, and only miR-1a-2 precursor was expressed in neonatal C57BL / 6 mouse cardiac fibroblasts at a similar level to that in neonatal C57BL / 6 mouse cardiomyocytes. miR-16-1 precursor was also similarly expressed in neonatal C57BL / 6 mouse cardiomyocytes and cardiac fibroblasts, however, miR-16-2 precursor was not shown to be expressed in either of these two kinds of neonatal C57BL / 6 mouse cardiac cells. miR-208a and miR-208b precursor was not found differentially expressed in neonatal C57BL / 6 mouse cardiomyocyte. Only miR-208a precursor was expressed in neonatal C57BL / 6 mouse cardiac fibroblasts at a level lower than that in neonatal C57BL / 6 mouse cardiomyocytes （P 0.05）. Conclusion The expression of miR-1, miR-16 and miR-208 precursor in neonatal mouse and rat cardiomyocytes and cardiac fibroblasts is cell type-specific.