摘要：
AIM:To investigate the regulation ofβ-adrenergic receptor (β-AR) agonist isoproterenol (ISO) on cardiac microRNA-21 (miR-21) expression.METHODS:The primary cultured mouse cardiomyocytes and cardiac fibroblasts were isolated by enzyme digestion and treated with ISO at 10μmol/L for 1, 6, 12, 24 and 48 h.The expression of miR-21 was detected by real-time PCR.The protein levels of p-STAT3 and STAT3 were determined by Western blot, and the concentration of interleukin-6 (IL-6) in the cultured supernatant was measured by ELISA.The cells were transfected with the luciferase reporter gene plasmid p GL3-21PPR containing the miR-21 promoter region, and the luciferase reporter gene assay was used to examine the effect of conditioned medium on the transcriptional activity of miR-21.RESULTS:The medium supernatant produced by ISO on cardiac fibroblasts was used as the conditioned medium, which increased the miR-21 expression in the cardiomyocytes in a time-dependent manner after fibroblasts was treated with ISO (P<0.05).The conditioned medium caused a significant increase in the transcriptional activity of miR-21 in the cardiomyocytes, while24 h and 48 h conditioned medium increased the transcriptional activity by 94.9％and 77.1％, respectively (P<0.01).The concentration of IL-6 in the conditioned medium was significantly increased, and the activity of transcriptional factor STAT3 was enhanced by paracrine action of IL-6 in the cardiomyocytes, which promoted the transcription and expression of miR-21.CONCLUSION:β-AR stimulation induces fibroblast synthesis and expression of IL-6 with paracrine effect on cardiomyocytes, up-regulates the expression of miR-21 in cardiomyocytes by IL-6/STAT3 pathway, and participates in the cardiac remodeling.%目的:探讨β-肾上腺素受体 (β-adrenergic receptor, β-AR) 激动剂异丙基肾上腺素 (isoproterenol, ISO) 促进心脏微小RNA-21 (miR-21) 表达的调控机制.方法:采用酶消化法分离原代乳小鼠心肌细胞和心脏成纤维细胞, 分别给予ISO (10μmol/L) 处理1、6、12、24和48 h, 采用real-time PCR法检测细胞miR-21表达水平, 采用Western blot法检测细胞p-STAT3和STAT3的蛋白水平, 采用ELISA法检测细胞上清中白细胞介素6 (IL-6) 的浓度.给予细胞转染含miR-21启动子区萤光素酶报告基因质粒p GL3-21PPR, 采用萤光素酶报告基因实验检测条件培养液对miR-21的转录活性的影响.结果:以ISO作用于心脏成纤维细胞产生的培养液上清作为条件培养液, 其可使心肌细胞miR-21的表达量随ISO作用于成纤维细胞时间的延长而逐渐增高 (P <0.05) ;该条件培养液可引起心肌细胞miR-21的转录活性显著增加, 其中ISO作用24 h和48 h的培养液分别使其转录活性增加94.9％和77.1％ (P <0.01) ;ISO作用成纤维细胞形成的条件培养液中IL-6浓度显著增加, 通过旁分泌作用于心肌细胞, 引起转录因子STAT3活性增强, 促进了miR-21的转录和表达.结论:激动β-AR可介导成纤维细胞合成和表达IL-6, 旁分泌作用于心肌细胞, 通过IL-6/STAT3途径, 上调心脏miR-21表达水平, 参与心脏重构.
