摘要
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。
AIM:To investigate the regulation of β-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 10 μmol/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 pGL3-21PPR 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,while 24 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.
作者
于海奕
宋峣
马晓伟
冯伟
高炜
张幼怡
YU Hai-yi;SONG Yao;MA Xiao-wei;FENG Wei;GAO Wei;ZHANG You-yi(Department of Cardiology and Institute of Vascular Medicine,Peking University Third Hospital,NHC Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides,Key Laboratory of Molecular Cardiovascular Science,Ministry of Education,Beijing Key Laboratory of Cardiovascular Receptors Research,Beijing 100191,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第2期193-199,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81530009
No.81670205
No.81871850)
北京市自然科学基金资助项目(No.7182176)