MiR-320a通过靶向抑制VEGF信号通路介导阿霉素心脏损伤
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摘要
目的探讨miR-320a在阿霉素心脏损伤中的作用与机制。方法在体实验中,我们在C57BL6小鼠上建立了阿霉素心脏损伤的动物模型。在造模之前应用重组腺相关病毒转导系统(rAAVs)来调控miR-320a在小鼠体内的表达,干预阿霉素后,通过Real-time PCR检测miR-320a的水平,通过血流动力学、心脏超声和形态学等方法检测各组小鼠心功能和心肌细胞面积。体外实验中,通过转染miR-320a mimics或inhibitor调控能内皮细胞miR-320a表达,检测内皮细胞增殖、凋亡、迁移和成管。结果阿霉素使心脏组织的miR-320a显著上调。高表达miR-320a加重了阿霉素引起的心功能恶化以及心肌细胞肥大。在体外实验中我们发现高表达miR-320a能抑制内皮细胞增殖,促进凋亡,使其迁移及成管能力下降,而采用miRNA inhibitor降低miR-320a表达则能减少阿霉素引起的损伤。同时我们证实VEGF-A是miR-320a的靶点。在体内腺相关病毒靶点回输实验中,在高表达miR-320a的基础上高表达VEGF-A减轻了miR-320a高表达造成的阿霉素对心脏心功能的损伤作用。结论 miR-320a通过调控内皮细胞功能介导了阿霉素的心脏损伤作用,抑制miR-320a具有缓解阿霉素心脏损伤的临床价值。
Objective To explore the role of miR-320a in doxorubicin induced cardiotoxicity. Methods Firstly, animal models of doxorubicin induced cardiotoxicity were established in C57 BL/6 mice. Real-time PCR were used to detect miR-320a level. Secondly, recombinant adenoassociated virus(rAAV) in vivo and miR-320a mimics/inhibitors in vitro were used to further explore the roles of miR-320a in doxorubicin induced cardiotoxicity. Lastly, target prediction showed that VEGF-A was a potential target of miR-320a, which was verified by anti-Ago2 coimmunoprecipitation and Western blots. To verify the function of VEGF-A in doxorubicin induced impairment, VEGF-A was over-expressed by rAAV in mice. Results Significantly increased level of miR-320a was found by after doxorubicin treatment, which was relative specificity in heart and endothelial cells. Knockdown of miR-320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR-320a enhanced apoptosis in vitro, and aggravated subsequent cardiac dysfunction in mice, while knockdown miR-320a or re-expression of VEGF-A eliminated the destructive effects of miR-320 in doxorubicin induced cardiotoxicity. Conclusion MiR-320a mediates doxorubicin induced cardiotoxicity by targeting VEGF signal pathway and thus,inhibition of miR-320a may be applied to the treatment of cardiac dysfunction induced by anthracycline.
Objective To explore the role of miR-320a in doxorubicin induced cardiotoxicity. Methods Firstly, animal models of doxorubicin induced cardiotoxicity were established in C57 BL/6 mice. Real-time PCR were used to detect miR-320a level. Secondly, recombinant adenoassociated virus(rAAV) in vivo and miR-320a mimics/inhibitors in vitro were used to further explore the roles of miR-320a in doxorubicin induced cardiotoxicity. Lastly, target prediction showed that VEGF-A was a potential target of miR-320a, which was verified by anti-Ago2 coimmunoprecipitation and Western blots. To verify the function of VEGF-A in doxorubicin induced impairment, VEGF-A was over-expressed by rAAV in mice. Results Significantly increased level of miR-320a was found by after doxorubicin treatment, which was relative specificity in heart and endothelial cells. Knockdown of miR-320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR-320a enhanced apoptosis in vitro, and aggravated subsequent cardiac dysfunction in mice, while knockdown miR-320a or re-expression of VEGF-A eliminated the destructive effects of miR-320 in doxorubicin induced cardiotoxicity. Conclusion MiR-320a mediates doxorubicin induced cardiotoxicity by targeting VEGF signal pathway and thus,inhibition of miR-320a may be applied to the treatment of cardiac dysfunction induced by anthracycline.
引文
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[15]Zampetaki A, Willeit P, Burr S. et al. Angiogenic microRNAs Linked to Incidence and Progression of Diabetic Retinopathy in Type 1Diabetes. Diabetes, 2016,65(1):216-227.
[16]Zhang S, Liu X, Bawa-Khalfe T, et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nature Med, 2012, 18(11):1639-1642.
[17]Zhu W, Zhang W, Shou W, et al. P53 inhibition exacerbates late-stage anthracycline cardiotoxicity. Cardiovasc Res, 2014,103(1):81-89.
[18]Bronisz A, Godlewski J, Wallace JA, et al. Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.Nature Cell Biology, 2012,14(2):159-167.
[19]Sato S, Katsushima K, Shinjo K, et al.Histone deacetylase inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer. Cancer Research, 2016,76:4192-4204.
[20]Feng B, Chakrabarti S. miR-320 Regulates Glucose-Induced Gene Expression in Diabetes. ISRNEndocrinol, 2012,2012:549875.
[21]Schaar DG, Medina DJ, Moore DF, et al. miR-320 targets transferrin receptor 1(CD71)and inhibits cell proliferation. Exp Hematol, 2009,37(2):245-255.
[22]Song CL, Liu B, Diao HY, et al. Down-regulation of microRNA-320suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1. Oncotarget, 2016,7(26):39740-39757.
[23]Duan H, Jiang Y, Zhang H, et al. MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene. Toxicol In Vitro, 2010,24(3):928-935.
[24]Hamam D, Ali D, Vishnubalaji R, et al. microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal(skeletal)stem cells. Cell Death Dis, 2014,5:e1499.
[25]Sun YH, Zhang ZH, Zhang HB, et al. miR-320 inhibits human coronary artery smooth muscle cell proliferation and migration by targeting Kruppel-like factor 5. Int J Clin Exp Patho, 2017, 10(3):2870-2878.
[26]Tang H, Lee M, Sharpe O, et al. Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.FASEBJ, 2012,26(11):4710-4721.
[2]Moslehi J, Cheng S. Cardio-oncology:it takes two to translate. Sci TranslMed, 2013,5(187):187fsl20.
[3]Carvalho FS, Burgeiro A, Garcia R, et al. Doxorubicin-induced cardiotoxicity:from bioenergetic failure and cell death to cardiomyopathy. Med Res Rev, 2014,34(1):106-135.
[4]Marti CN, Gheorghiade M, Kalogeropoulos AP, et al. Endothelial dysfunction, arterial stiffness, and heart failure. J Am Coll Cardiol,2012,60(16):1455-1469.
[5]Liu S, Premont RT, Rockey DC. G-protein-coupled receptor kinase interactor-1(GIT1)is a new endothelial nitric-oxide synthase(eNOS)interactor with functional effects on vascular homeostasis. J Biol Chem, 2012,287(15):12309-12320.
[6]Moyes AJ, Khambata RS, Villar I, et al. Endothelial C-type natriuretic peptide maintains vascular homeostasis. J Clin Invest, 2014, 124(9):4039-4051.
[7]Tanaka T, Yamaguchi J, Shoji K, et al. Anthracycline inhibits recruitment of hypoxia-inducible transcription factors and suppresses tumor cell migration and cardiac angiogenic response in the host. J Biol Chem, 2012,287(42):34866-34882.
[8]Wu S, Ko YS, Teng MS, et al. Adriamycin-induced cardiomyocyte and endothelial cell apoptosis:in vitro and in vivo studies. J Mol Cell Cardiol, 2002,34(12):1595-1607.
[9]Taimeh Z, Loughran J, Birks EJ, et al. Vascular endothelial growth factor in heart failure. NatRevi Cardiol, 2013,10(9):519-530.
[10]Conomopoulou P, Kotsakis A, Kapiris I, et al. Cancer therapy and cardiovascular risk:focus on bevacizumab. Cancer Manag Res, 2015,7:133-143.
[11]Flood EC, Hajjar KA. The annexin A2 system and vascular homeostasis. Vascul Pharmacol, 2011, 54(3-6):59-67.
[12]Chen C, Wang Y, Yang S, et al. MiR-320a contributes to atherogenesis by augmenting multiple risk factors and down-regulating SRF. J Cell Mol Med, 2015,19(5):970-985.
[13]Wang XH, Qian RZ, Zhang W, et al. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats. Clin Exp Pharmacol Physiol, 2009, 36(2):181-188.
[14]Ren XP. Wu JH, Wang XH, et al. MicroRNA-320 Is Involved in the Regulation of Cardiac Ischemia/Reperfusion Injury by Targeting Heat-Shock Protein 20. Circulation, 2009,119(17):2357-U2128.
[15]Zampetaki A, Willeit P, Burr S. et al. Angiogenic microRNAs Linked to Incidence and Progression of Diabetic Retinopathy in Type 1Diabetes. Diabetes, 2016,65(1):216-227.
[16]Zhang S, Liu X, Bawa-Khalfe T, et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nature Med, 2012, 18(11):1639-1642.
[17]Zhu W, Zhang W, Shou W, et al. P53 inhibition exacerbates late-stage anthracycline cardiotoxicity. Cardiovasc Res, 2014,103(1):81-89.
[18]Bronisz A, Godlewski J, Wallace JA, et al. Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.Nature Cell Biology, 2012,14(2):159-167.
[19]Sato S, Katsushima K, Shinjo K, et al.Histone deacetylase inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer. Cancer Research, 2016,76:4192-4204.
[20]Feng B, Chakrabarti S. miR-320 Regulates Glucose-Induced Gene Expression in Diabetes. ISRNEndocrinol, 2012,2012:549875.
[21]Schaar DG, Medina DJ, Moore DF, et al. miR-320 targets transferrin receptor 1(CD71)and inhibits cell proliferation. Exp Hematol, 2009,37(2):245-255.
[22]Song CL, Liu B, Diao HY, et al. Down-regulation of microRNA-320suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1. Oncotarget, 2016,7(26):39740-39757.
[23]Duan H, Jiang Y, Zhang H, et al. MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene. Toxicol In Vitro, 2010,24(3):928-935.
[24]Hamam D, Ali D, Vishnubalaji R, et al. microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal(skeletal)stem cells. Cell Death Dis, 2014,5:e1499.
[25]Sun YH, Zhang ZH, Zhang HB, et al. miR-320 inhibits human coronary artery smooth muscle cell proliferation and migration by targeting Kruppel-like factor 5. Int J Clin Exp Patho, 2017, 10(3):2870-2878.
[26]Tang H, Lee M, Sharpe O, et al. Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.FASEBJ, 2012,26(11):4710-4721.