β-肾上腺素受体介导成纤维细胞旁分泌IL-6促进心脏miR-21表达
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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 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.
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.
引文
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[21]Li Y,Zhang H,Liao W,et al.Transactivated EGFR mediatesα1-AR-induced STAT3 activation and cardiac hypertrophy[J].Am J Physiol Heart Circ Physiol,2011,301(5):H1941-H1951.
[22]Zhang H,Feng W,Liao W,et al.The gp130/STAT3 signaling pathway mediates beta-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes[J].FEBS J,2008,275(14):3590-3597.
[23]Yin F,Li P,Zheng M,et al.Interleukin-6 family of cytokines mediates isoproterenol-induced delayed STAT3 activation in mouse heart[J].J Biol Chem,2003,278(23):21070-21075.
[24]Loffler D,Brocke-Heidrich K,Pfeifer G,et al.Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21through a highly conserved enhancer[J].Blood,2007,110(4):1330-1333.
[25]Bageghni SA,Hemmings KE,Zava N,et al.Cardiac fibroblast-specific p38αMAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism[J].FASEB J,2018,32(9):4941-4954.
[2]de Lucia C,Eguchi A,Koch WJ.New insights in cardiac beta-adrenergic signaling during heart failure and aging[J].Front Pharmacol,2018,9:904.
[3]Wang J,Gareri C,Rockman HA.G-protein-coupled receptors in heart disease[J].Circ Res,2018,123(6):716-735.
[4]Ma X,Song Y,Chen C,et al.Distinct actions of intermittent and sustained beta-adrenoceptor stimulation on cardiac remodeling[J].Sci China Life Sci,2011,54(6):493-501.
[5]Fu Y,Xiao H,Zhang Y.Beta-adrenoceptor signaling pathways mediate cardiac pathological remodeling[J].Front Biosci(Elite Ed),2012,4:1625-1637.
[6]Chen C,Ponnusamy M,Liu C,et al.MicroRNA as a therapeutic target in cardiac remodeling[J].Biomed Res Int,2017,2017:1278436.
[7]Sun M,Yu H,Zhang Y,et al.MicroRNA-214 mediates isoproterenol-induced proliferation and collagen synthesis in cardiac fibroblasts[J].Sci Rep,2015,5:18351.
[8]Hou Y,Sun Y,Shan H,et al.beta-adrenoceptor regulates miRNA expression in rat heart[J].Med Sci Monit,2012,18(8):R309-R314.
[9]Tatsuguchi M,Seok HY,Callis TE,et al.Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy[J].J Mol Cell Cardiol,2007,42(6):1137-1141.
[10]Cheng Y,Zhang C.MicroRNA-21 in cardiovascular disease[J].J Cardiovasc Transl Res,2010,3(3):251-255.
[11]Liu Y,Nie H,Zhang K,et al.A feedback regulatory loop between HIF-1alpha and miR-21 in response to hypoxia in cardiomyocytes[J].FEBS Lett,2014,588(17):3137-3146.
[12]Gutsaeva DR,Thounaojam M,Rajpurohit S,et al.STAT3-mediated activation of miR-21 is involved in downregulation of TIMP3 and neovascularization in the ischemic retina[J].Oncotarget,2017,8(61):103568-103580.
[13]毛彦稳,张小欢,彭伟.α-硫辛酸对糖尿病大鼠肾脏miR-21和Smad7表达及纤维化的影响[J].中国病理生理杂志,2018,34(10):1843-1847.
[14]Frankel LB,Christoffersen NR,Jacobsen A,et al.Programmed cell death 4(PDCD4)is an important functional target of the microRNA miR-21 in breast cancer cells[J].J Biol Chem,2008,283(2):1026-1033.
[15]van Rooij E,Sutherland LB,Liu N,et al.A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure[J].Proc Natl Acad Sci U S A,2006,103(48):18255-18260.
[16]Sayed D,Hong C,Chen IY,et al.MicroRNAs play an essential role in the development of cardiac hypertrophy[J].Circ Res,2007,100(3):416-424.
[17]吴美华,曾建斌,熊向阳.血浆microRNA-21水平与左室射血分数保留的心力衰竭相关性肺高压的关系[J].中国病理生理杂志,2018,34(10):1784-1789.
[18]Sayed D,Rane S,Lypowy J,et al.MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths[J].Mol Biol Cell,2008,19(8):3272-3282.
[19]Krichevsky AM,Gabriely G.miR-21:a small multi-faceted RNA[J].J Cell Mol Med,2009,13(1):39-53.
[20]Choy MK,Movassagh M,Siggens L,et al.High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-κB-complex-dependent gene expression in human heart failure[J].Genome Med,2010,2(6):37.
[21]Li Y,Zhang H,Liao W,et al.Transactivated EGFR mediatesα1-AR-induced STAT3 activation and cardiac hypertrophy[J].Am J Physiol Heart Circ Physiol,2011,301(5):H1941-H1951.
[22]Zhang H,Feng W,Liao W,et al.The gp130/STAT3 signaling pathway mediates beta-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes[J].FEBS J,2008,275(14):3590-3597.
[23]Yin F,Li P,Zheng M,et al.Interleukin-6 family of cytokines mediates isoproterenol-induced delayed STAT3 activation in mouse heart[J].J Biol Chem,2003,278(23):21070-21075.
[24]Loffler D,Brocke-Heidrich K,Pfeifer G,et al.Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21through a highly conserved enhancer[J].Blood,2007,110(4):1330-1333.
[25]Bageghni SA,Hemmings KE,Zava N,et al.Cardiac fibroblast-specific p38αMAP kinase promotes cardiac hypertrophy via a putative paracrine interleukin-6 signaling mechanism[J].FASEB J,2018,32(9):4941-4954.