微小RNA-23b-3p通过靶向TGFBR3促进人心房肌成纤维细胞纤维化相关基因表达
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摘要
目的:探究微小RNA-23b-3p(mi R-23b-3p)对人心房肌成纤维细胞中纤维化相关基因表达的作用及其可能作用靶基因。方法:分离并体外培养房颤患者心耳中原代心房肌成纤维细胞,并用细胞免疫荧光染色实验鉴定;双萤光素酶报告基因实验检测mi R-23b-3p与潜在靶基因转化生长因子β受体3(TGFBR3) 3'端非翻译区(3'-UTR)的结合作用; CCK-8、Ed U染色及Transwell实验检测细胞活力、增殖及迁移能力,RT-qPCR和Western blot法检测TGFBR3及纤维化相关基因的m RNA和蛋白表达。结果:在人心房肌成纤维细胞中过表达mi R-23b-3p不影响细胞的活力、增殖及迁移能力,但可显著增强细胞中纤维化相关基因COL1A1、COL3A1和ACTA2的表达(P <0. 05或P <0. 01)。双萤光素酶报告基因实验显示mi R-23b-3p与TGFBR3 3'-UTR有结合作用。RT-qPCR和Western blot结果证实mi R-23b-3p可在转录水平抑制TGFBR3表达。过表达mi R-23b-3p和沉默TGFBR3均能显著促进人心房肌成纤维细胞中Smad3激活和纤维化相关基因表达(P <0. 05或P <0. 01)。结论:TGFBR3是mi R-23b-3p的作用靶基因,并介导mi R-23b-3p促进心房肌成纤维细胞中纤维化相关基因表达。
AIM: To investigate the role of micro RNA-23b-3p( mi R-23b-3p) in myocardial fibrosis and the potential target of mi R-23b-3p. METHODS: Human atrial fibroblasts( HAFs) were isolated and cultured in vitro for the cellular experimental study. HAFs were identified by ACTA2 immunofluorescence staining. Dual-luciferase reporter assay was performed to confirm the interaction between mi R-23b-3p and the 3'-UTR of transforming growth factor β receptor 3( TGFBR3). CCK-8,Ed U and Transwell assays were used to detect the viability,proliferation and migration of HAFs with mi R-23b-3p overexpression. The m RNA and protein expression levels of TGFBR3 and fibrosis-related genes were determined by RT-qPCR and Western blot,respectively. RESULTS: Immunofluorescence staining showed that the expression of ACTA2 was positive in the isolated HAFs. Overexpression of mi R-23b-3p significantly enhanced the expression of fibrosis-related genes genes COL1A1,COL3A1 and ACTA2( P < 0. 05 or P < 0. 01),with no effects on the viability,proliferation and migration of HAFs. Moreover,the dual-luciferase reporter assay revealed that mi R-23b-3p interacted with the 3'-UTR of TGFBR3. mi R-23b-3p was observed to inhibit TGFBR3 expression at the transcriptional level. Meanwhile,mi R-23b-3p mimic,in parallel to TGFBR3 si RNA,inhibited TGFBR3 expression,and markedly enhanced Smad3 activation and the expression of fibrosis-related genes in the HAFs( P < 0. 05 or P < 0. 01). CONCLUSION: TGFBR3 is a target gene of mi R-23b-3p,and mediates the pro-fibrotic effect of mi R-23b-3p in the HAFs.
AIM: To investigate the role of micro RNA-23b-3p( mi R-23b-3p) in myocardial fibrosis and the potential target of mi R-23b-3p. METHODS: Human atrial fibroblasts( HAFs) were isolated and cultured in vitro for the cellular experimental study. HAFs were identified by ACTA2 immunofluorescence staining. Dual-luciferase reporter assay was performed to confirm the interaction between mi R-23b-3p and the 3'-UTR of transforming growth factor β receptor 3( TGFBR3). CCK-8,Ed U and Transwell assays were used to detect the viability,proliferation and migration of HAFs with mi R-23b-3p overexpression. The m RNA and protein expression levels of TGFBR3 and fibrosis-related genes were determined by RT-qPCR and Western blot,respectively. RESULTS: Immunofluorescence staining showed that the expression of ACTA2 was positive in the isolated HAFs. Overexpression of mi R-23b-3p significantly enhanced the expression of fibrosis-related genes genes COL1A1,COL3A1 and ACTA2( P < 0. 05 or P < 0. 01),with no effects on the viability,proliferation and migration of HAFs. Moreover,the dual-luciferase reporter assay revealed that mi R-23b-3p interacted with the 3'-UTR of TGFBR3. mi R-23b-3p was observed to inhibit TGFBR3 expression at the transcriptional level. Meanwhile,mi R-23b-3p mimic,in parallel to TGFBR3 si RNA,inhibited TGFBR3 expression,and markedly enhanced Smad3 activation and the expression of fibrosis-related genes in the HAFs( P < 0. 05 or P < 0. 01). CONCLUSION: TGFBR3 is a target gene of mi R-23b-3p,and mediates the pro-fibrotic effect of mi R-23b-3p in the HAFs.
引文
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[21]Groeneveld ME,Bogunovic N,RJP M,et al.Betaglycan(TGFBR3)up-regulation correlates with increased TGF-βsignaling in Marfan patient fibroblasts in vitro[J].Cardiovasc Pathol,2018,32:44-49.
[22]Chen Y,Yang P,Li F,et al.Association analysis of TGFBR3 gene with Vogt-Koyanagi-Harada disease and Behcet's disease in the Chinese Han population[J].Curr Eye Res,2012,37(4):312-317.
[23]Liang H,Zhang C,Ban T,et al.A novel reciprocal loop between micro RNA-21 and TGFβRIII is involved in cardiac fibrosis[J].Int J Biochem Cell Biol,2012,44(12):2152-2160.
[24]Ahn JY,Park S,Yun YS,et al.Inhibition of type IIITGF-βreceptor aggravates lung fibrotic process[J].Biomed Pharmacother,2010,64(7):472-476.
[25]Hermida N,López B,González A,et al.A synthetic peptide from transforming growth factor-β1 type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats[J].Cardiovasc Res,2009,81(3):601-609.
[26]Chu W,Li X,Li C,et al.TGFBR3,a potential negative regulator of TGF-βsignaling,protects cardiac fibroblasts from hypoxia-induced apoptosis[J].J Cell Physiol,2011,226(10):2586-2594.
[2]Heijman J,Algalarrondo V,Voigt N,et al.The value of basic research insights into atrial fibrillation mechanisms as a guide to therapeutic innovation:a critical analysis[J].Cardiovasc Res,2016,109(4):467-479.
[3]Jalife J,Kaur K.Atrial remodeling,fibrosis,and atrial fibrillation[J].Trends Cardiovasc Med,2015,25(6):475-484.
[4]Zahid S,Cochet H,Boyle PM,et al.Patient-derived models link re-entrant driver localization in atrial fibrillation to fibrosis spatial pattern[J].Cardiovasc Res,2016,110(3):443-454.
[5]Gal P,Marrouche NF.Magnetic resonance imaging of atrial fibrosis:redefining atrial fibrillation to a syndrome[J].Eur Heart J,2017,38(1):14-19.
[6]Novitskaya T,Chepurko E,Covarrubias R,et al.Extracellular nucleotide regulation and signaling in cardiac fibrosis[J].J Mol Cell Cardiol,2016,93:47-56.
[7]Ambros V.The functions of animal micro RNAs[J].Nature,2004,431(7006):350-355.
[8]Kim VN.MicroRNA biogenesis:coordinated cropping and dicing[J].Nat Rev Mol Cell Biol,2005,6(5):376-385.
[9]Cao W,Shi P,Ge JJ.mi R-21 enhances cardiac fibrotic remodeling and fibroblast proliferation via CADM1/STAT3pathway[J].BMC Cardiovasc Disord,2017,17:88.
[10]Adam O,L9hfelm B,Thum T,et al.Role of mi R-21 in the pathogenesis of atrial fibrosis[J].Basic Res Cardiol,2012,107(5):278.
[11]Li H,Li S,Yu B,et al.Expression of mi R-133 and mi R-30 in chronic atrial fibrillation in canines[J].Mol Med Rep,2012,5(6):1457-1460.
[12]Yang B,Lin H,Xiao J,et al.The muscle-specific micro RNA mi R-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2[J].Nat Med,2007,13(4):486-491.
[13]Shan ZX,Lin QX,Deng CY,et al.mi R-1/mi R-206 regulate Hsp60 expression contributing to glucose-mediated apoptosis in cardiomyocytes[J].FEBS Lett,2010,584(16):3592-3600.
[14]Zhang H,Caudle Y,Shaikh A,et al.Inhibition of micro RNA-23b prevents polymicrobial sepsis-induced cardiac dysfunction by modulating TGIF1 and PTEN[J].Biomed Pharmacother,2018,103:869-878.
[15]Pandey R,Yang Y,Jackson L,et al.MicroRNAs regulating Meis1 expression and inducing cardiomyocyte proliferation[J].Cardiovasc Regen Med,2016,3:e1468.
[16]Tao H,Shi KH,Yang JJ,et al.Epigenetic regulation of cardiac fibrosis[J].Cell Signal,2013,25(9):1932-1938.
[17]Tao H,Chen ZW,Yang JJ,et al.MicroRNA-29a suppresses cardiac fibroblasts proliferation via targeting VEGF-A/MAPK signal pathway[J].Int J Biol Macromol,2016,88:414-423.
[18]张灼,朱杰宁,肖珍,等.微小RNA-199a-5p通过靶向SIRT1促进心肌纤维化相关基因表达[J].中国病理生理杂志,2017,33(10):1781-1787.
[19]Wang J,Huang W,Xu R,et al.MicroRNA-24 regulates cardiac fibrosis after myocardial infarction[J].J Cell Mol Med,2012,16(9):2150-2160.
[20]Schwartze JT,Becker S,Sakkas E,et al.Glucocorticoids recruit Tgfbr3 and Smad1 to shift transforming growth factor-βsignaling from the Tgfbr1/Smad2/3 axis to the Acvrl1/Smad1 axis in lung fibroblasts[J].J Biol Chem,2014,289(6):3262-3275.
[21]Groeneveld ME,Bogunovic N,RJP M,et al.Betaglycan(TGFBR3)up-regulation correlates with increased TGF-βsignaling in Marfan patient fibroblasts in vitro[J].Cardiovasc Pathol,2018,32:44-49.
[22]Chen Y,Yang P,Li F,et al.Association analysis of TGFBR3 gene with Vogt-Koyanagi-Harada disease and Behcet's disease in the Chinese Han population[J].Curr Eye Res,2012,37(4):312-317.
[23]Liang H,Zhang C,Ban T,et al.A novel reciprocal loop between micro RNA-21 and TGFβRIII is involved in cardiac fibrosis[J].Int J Biochem Cell Biol,2012,44(12):2152-2160.
[24]Ahn JY,Park S,Yun YS,et al.Inhibition of type IIITGF-βreceptor aggravates lung fibrotic process[J].Biomed Pharmacother,2010,64(7):472-476.
[25]Hermida N,López B,González A,et al.A synthetic peptide from transforming growth factor-β1 type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats[J].Cardiovasc Res,2009,81(3):601-609.
[26]Chu W,Li X,Li C,et al.TGFBR3,a potential negative regulator of TGF-βsignaling,protects cardiac fibroblasts from hypoxia-induced apoptosis[J].J Cell Physiol,2011,226(10):2586-2594.
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