Lin28B/let-7d环路调控成纤维细胞功能参与肺纤维化发生
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摘要
目的研究Lin28B/let-7d环路诱导肺成纤维细胞增殖、分化的作用及分子机制,为临床肺纤维化的治疗提供新的策略。方法气管注射博来霉素(bleomycin,BLM)造成小鼠肺纤维化模型;血管紧张素Ⅱ(AngⅡ)及转化生长因子(transforming growth factor,TGF)-β1诱导人胚肺成纤维细胞(MRC-5)纤维性变;qRT-PCR检测组织及细胞中Lin28B、collagen 1α1、collagen 3α1变化;Western blot检测Lin28B蛋白表达;MTT、Edu染色和免疫荧光实验分别检测细胞活力、增殖及成纤维细胞向肌成纤维细胞的转变。结果 Lin28B在肺纤维化小鼠和细胞中表达明显升高。Lin28B可诱导MRC-5细胞胶原合成增加,而过表达let-7d则减轻Lin28B的这一作用。进一步研究发现,Lin28B可诱导成纤维细胞增殖,并促进成纤维细胞向肌成纤维细胞转变,这一作用是通过抑制let-7d来实现的。结论 Lin-28B通过抑制let-7d进而促进成纤维细胞增殖、分化,最终增加成纤维细胞胶原合成,诱发肺纤维化的发生,Lin28B可能作为肺纤维化的治疗新靶点。
Aim To examine the role and uderlying mechanisms of Lin28/let-7d axis in the proliferation of lung fibrobalsts and fibroblasts-into-myfibroblasts transition,and provide novel strategy for the treatment of idiopathic pulmonary fibrosis( IPF). Methods We induced experimental lung fibrosis in mice by intratracheally injection of bleomycin( BLM). Ang Ⅱ and TGF-β1 were used to induce fibrogenesis in cultured MRC-5 cells;qRT-PCR and Western blot were applied to determine the changes of Lin28 B,collagen 1α1 and collagen 3α1; MTT assay,Edu satining and immunofluoresence were used to examine the cell viability,proliferation and fibroblasts-into-myofibroblasts transition in MRC-5 cells. Results Lin28 B was increased in the lung of mice with experimental lung fibrosis and in MRC-5 cells treated with AngⅡ or TGF-β1. Moreover,Lin28 B enhanced collagen deposition via inhibiting expression of let-7d,which maybe contribute to the progression of IPF. In addition, further studies showed that Lin28 B promoted proliferation and fibroblasts-into-myofibroblasts in MRC-5 cells. Conclusion Lin28B/let-7d axis contributes to fibrogenesis via promotes fibroblasts-into-myofibroblasts transition,which may provide novel approaches for lung fibrosis treatment.
Aim To examine the role and uderlying mechanisms of Lin28/let-7d axis in the proliferation of lung fibrobalsts and fibroblasts-into-myfibroblasts transition,and provide novel strategy for the treatment of idiopathic pulmonary fibrosis( IPF). Methods We induced experimental lung fibrosis in mice by intratracheally injection of bleomycin( BLM). Ang Ⅱ and TGF-β1 were used to induce fibrogenesis in cultured MRC-5 cells;qRT-PCR and Western blot were applied to determine the changes of Lin28 B,collagen 1α1 and collagen 3α1; MTT assay,Edu satining and immunofluoresence were used to examine the cell viability,proliferation and fibroblasts-into-myofibroblasts transition in MRC-5 cells. Results Lin28 B was increased in the lung of mice with experimental lung fibrosis and in MRC-5 cells treated with AngⅡ or TGF-β1. Moreover,Lin28 B enhanced collagen deposition via inhibiting expression of let-7d,which maybe contribute to the progression of IPF. In addition, further studies showed that Lin28 B promoted proliferation and fibroblasts-into-myofibroblasts in MRC-5 cells. Conclusion Lin28B/let-7d axis contributes to fibrogenesis via promotes fibroblasts-into-myofibroblasts transition,which may provide novel approaches for lung fibrosis treatment.
引文
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[12]Takashima Y,Terada M,Udono M,et al.Suppression of lethal-7b and miR-125a/b maturation by Lin28b enables maintenance of stem cell properties in hepatoblasts[J].Hepatology,2016,64(1):245-60.
[13]Liang H,Gu Y,Li T,et al.Integrated analyses identify the involvement of microRNA-26a in epithelial-mesenchymal transition during idiopathic pulmonary fibrosis[J].Cell Death Dis,2014,5:e1238.
[14]Fierro-Fernandez M,Busnadiego O,Sandoval P,et al.miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2[J].EMBO Rep,2015,16(10):1358-77.
[15]Liu G,Friggeri A,Yang Y,et al.miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis[J].J Exp Med,2010,207(8):1589-97.
[16]刘晓丽,王如峰,胡秀华.MicroRNAs在呼吸系统疾病预防和治疗中的研究进展[J].中国药理学通报,2015,31(7):895-9.[16]Liu X L,Wang R F,Hu X H.Advanced study on microRNAs involved in prevention and treatment of respiratory system diseases[J].Chin Pharmacol Bull,2015,31(7):895-9.
[2]Ambros V.microRNAs:tiny regulators with great potential[J].Cell,2001,107(7):823-6.
[3]Pandit K V,Corcoran D,Yousef H,et al.Inhibition and role of let-7d in idiopathic pulmonary fibrosis[J].Am J Respir Crit Care Med,2010,182(2):220-9.
[4]Albino D,Civenni G,Dallavalle C,et al.Activation of the Lin28/let-7 axis by loss of ESE3/EHF promotes a tumorigenic and stem-like phenotype in prostate cancer[J].Cancer Res,2016,76(12):3629-43.
[5]Liang H,Liu S,Chen Y,et al.miR-26a suppresses EMT by disrupting the Lin28B/let-7d axis:potential cross-talks among miRNAs in IPF[J].J Mol Med(Berl),2016,94(6):655-65.
[6]Liang H,Xu C,Pan Z,et al.The antifibrotic effects and mechanisms of microRNA-26a action in idiopathic pulmonary fibrosis[J].Mol Ther,2014,22(6):1122-33.
[7]Hinz B,Phan S H,Thannickal V J,et al.The myofibroblast:one function,multiple origins[J].Am J Pathol,2007,170(6):1807-16.
[8]Huleihel L,Ben-Yehudah A,Milosevic J,et al.Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts[J].Am J Physiol Lung Cell Mol Physiol,2014,306(6):L534-42.
[9]Desjardins A,Bouvette J,Legault P.Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors[J].Nucleic Acids Res,2014,42(7):4615-28.
[10]Zhang J,Ratanasirintrawoot S,Chandrasekaran S,et al.Lin28regulates stem cell metabolism and conversion to primed pluripotency[J].Cell Stem Cell,2016,19(1):66-80.
[11]Mc Daniel K,Hall C,Sato K,et al.Lin28 and let-7:roles and regulation in liver diseases[J].Am J Physiol Gastrointest Liver Physiol,2016,310(10):G757-65.
[12]Takashima Y,Terada M,Udono M,et al.Suppression of lethal-7b and miR-125a/b maturation by Lin28b enables maintenance of stem cell properties in hepatoblasts[J].Hepatology,2016,64(1):245-60.
[13]Liang H,Gu Y,Li T,et al.Integrated analyses identify the involvement of microRNA-26a in epithelial-mesenchymal transition during idiopathic pulmonary fibrosis[J].Cell Death Dis,2014,5:e1238.
[14]Fierro-Fernandez M,Busnadiego O,Sandoval P,et al.miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2[J].EMBO Rep,2015,16(10):1358-77.
[15]Liu G,Friggeri A,Yang Y,et al.miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis[J].J Exp Med,2010,207(8):1589-97.
[16]刘晓丽,王如峰,胡秀华.MicroRNAs在呼吸系统疾病预防和治疗中的研究进展[J].中国药理学通报,2015,31(7):895-9.[16]Liu X L,Wang R F,Hu X H.Advanced study on microRNAs involved in prevention and treatment of respiratory system diseases[J].Chin Pharmacol Bull,2015,31(7):895-9.