miR-27a-3p通过Wnt3a/β-catenin信号通路抑制肺成纤维细胞I、III型胶原合成
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摘要
目的探讨microRNA-27a-3p(miR-27a-3p)对肺成纤维细胞I型胶原(ColⅠ)和III型胶原(ColⅢ)合成的影响及分子机制。方法培养人胚肺成纤维细胞MRC-5,转染miR-27a-3p模拟物/抑制物,实时荧光定量PCR(qPCR)检测miR-27a-3p水平;qPCR和Western blot测定ColⅠ、ColⅢ、Wnt3a表达;Western blot分析细胞核内β-catenin水平。生物信息学预测miR-27a-3p与Wnt3a 3′-非翻译区(3′-UTR)靶向结合情况,并用双荧光素酶报告基因检测miR-27a-3p模拟物对Wnt3a 3′-UTR野生型和突变型荧光素酶活性的影响。给予Wnt3a/β-catenin信号通路抑制剂Dkk1预处理MRC-5细胞6 h,再用miR-27a-3p抑制物转染细胞48 h,qPCR和Western blot检测ColⅠ、ColⅢ表达。结果 miR-27a-3p模拟物明显增加miR-27a-3p水平,降低ColⅠ、ColⅢ、Wnt3a和β-catenin表达,其抑制物的作用则相反,与对照组比较,差异均有显著性(P<0.01)。Wnt3a 3′-UTR存在1个miR-27a-3p的结合位点,miR-27a-3p过表达减少野生型Wnt3a 3′-UTR荧光素酶活性(P<0.01),但对其突变型荧光素酶活性无影响(P>0.05)。Dkk1预处理几乎完全逆转miR-27a-3p抑制物对ColⅠ、ColⅢ合成的诱导作用(P<0.05)。结论 miR-27a-3p抑制肺成纤维细胞ColⅠ、ColⅢ生物合成,其机制与拮抗Wnt3a/β-catenin信号通路有关。
Aim To explore the effects of microRNA-27 a-3 p(miR-27 a-3 p) on collagen type Ⅰ(Col Ⅰ) and collagen type Ⅲ(Col Ⅲ) synthesis in pulmonary fibroblasts and the underlying mechanisms.Methods Human pulmonary fibroblasts MRC-5 were cultured and then transfected with miR-27 a-3 p mimic or its inhibitor. qPCR and Western blot were used to detect miR-27 a-3 p levels and nuclear β-catenin content, respectively. The expression of Col Ⅰ, Col Ⅲ and Wnt3 a was measured using qPCR and Western blot. Bioinformatics predicted the potential of miR-27 a-3 p bound to Wnt3 a 3′-untranslated region(3′-UTR). Dual luciferase reporter gene analyzed the effects of miR-27 a-3 p mimic transfection on luciferase activity of wild type and mutant Wnt3 a 3′-UTR. MRC-5 cells were treated with the Wnt3 a/β-catenin signaling pathway inhibitor Dkk1, followed by transfection with miR-27 a-3 p mimic or its inhibitor. Col Ⅰ and Col Ⅲ expression was detected by qPCR and Western blot. Results miR-27 a-3 p mimic markedly increased miR-27 a-3 p levels and decreased Col Ⅰ, Col Ⅲ, Wnt3 a and β-catenin expression(P<0.01), while an opposite effect was observed in response to miR-27 a-3 p inhibitor(P<0.01). Bioinformatics prediction showed a binding site of miR-27 a-3 p in the 3′-UTR of Wnt3 a. Importantly, miR-27 a-3 p mimic reduced luciferase activity of wild type Wnt3 a 3′-UTR(P<0.01) but not its mutant(P>0.05), revealing Wnt3 a as a target of miR-27 a-3 p. In addition, Dkk1 pretreatment almost fully reversed the inductive effect of miR-27 a-3 p inhibitor on Col Ⅰ and Col Ⅲ expression in MRC-5 cells(P<0.05).Conclusions miR-27 a-3 p inhibits the biosynthesis of Col Ⅰ and Col Ⅲ in pulmonary fibroblasts, which is attributed to inactivated Wnt3 a/β-catenin signaling pathway.
Aim To explore the effects of microRNA-27 a-3 p(miR-27 a-3 p) on collagen type Ⅰ(Col Ⅰ) and collagen type Ⅲ(Col Ⅲ) synthesis in pulmonary fibroblasts and the underlying mechanisms.Methods Human pulmonary fibroblasts MRC-5 were cultured and then transfected with miR-27 a-3 p mimic or its inhibitor. qPCR and Western blot were used to detect miR-27 a-3 p levels and nuclear β-catenin content, respectively. The expression of Col Ⅰ, Col Ⅲ and Wnt3 a was measured using qPCR and Western blot. Bioinformatics predicted the potential of miR-27 a-3 p bound to Wnt3 a 3′-untranslated region(3′-UTR). Dual luciferase reporter gene analyzed the effects of miR-27 a-3 p mimic transfection on luciferase activity of wild type and mutant Wnt3 a 3′-UTR. MRC-5 cells were treated with the Wnt3 a/β-catenin signaling pathway inhibitor Dkk1, followed by transfection with miR-27 a-3 p mimic or its inhibitor. Col Ⅰ and Col Ⅲ expression was detected by qPCR and Western blot. Results miR-27 a-3 p mimic markedly increased miR-27 a-3 p levels and decreased Col Ⅰ, Col Ⅲ, Wnt3 a and β-catenin expression(P<0.01), while an opposite effect was observed in response to miR-27 a-3 p inhibitor(P<0.01). Bioinformatics prediction showed a binding site of miR-27 a-3 p in the 3′-UTR of Wnt3 a. Importantly, miR-27 a-3 p mimic reduced luciferase activity of wild type Wnt3 a 3′-UTR(P<0.01) but not its mutant(P>0.05), revealing Wnt3 a as a target of miR-27 a-3 p. In addition, Dkk1 pretreatment almost fully reversed the inductive effect of miR-27 a-3 p inhibitor on Col Ⅰ and Col Ⅲ expression in MRC-5 cells(P<0.05).Conclusions miR-27 a-3 p inhibits the biosynthesis of Col Ⅰ and Col Ⅲ in pulmonary fibroblasts, which is attributed to inactivated Wnt3 a/β-catenin signaling pathway.
引文
[1] George G, Vaid U, Summer R. Therapeutic advances in idiopathic pulmonary fibrosis[J]. Clin Pharmacol Ther, 2016, 99(1):30-2.
[2] 刘理静,钱红,尹辉明,等. IL-17单克隆抗体对肺纤维化大鼠的保护作用及部分机制研究[J].中国药理学通报, 2015, 31(11):1586-92. Liu L J, Qian H, Yin H M, et al. Protective effect of IL-17 monoclonal antibody on rats with pulmonary fibrosis and its partial mechanisms[J]. Chin Pharmacol Bull, 2015, 31(11):1586-92.
[3] Wang Y, Liu J, Chen J, et al. MiR-29 mediates TGFβ1-induced extracellular matrix synthesis through activation of Wnt/β-catenin pathway in human pulmonary fibroblasts[J]. Technol Health Care, 2015, 23(Suppl 1):S119-25.
[4] Tan J, Tong B D, Wu Y J, et al. MicroRNA-29 mediates TGFβ1-induced extracellular matrix synthesis by targeting Wnt/β-catenin pathway in human orbital fibroblasts[J]. Int J Clin Exp Pathol, 2014, 7(11):7571-7.
[5] 刘理静, 钱红. 上调 miR-21表达促进肺成纤维细胞增殖和胶原蛋白合成[J]. 细胞与分子免疫学杂志, 2015, 31(7):918-22. Liu L J, Qian H. Up-regulation of miR-21 promotes cell proliferation and collagen synthesis in pulmonary fibroblasts[J]. Chin J Cell Mol Immunol, 2015, 31(7):918-22.
[6] Liu B, Li R, Zhang J, et al. MicroRNA-708-3p as a potential therapeutic target via the ADAM17-GATA/STAT3 axis in idiopathic pulmonary fibrosis[J]. Exp Mol Med, 2018, 50(3):e465.
[7] Cui H, Banerjee S, Xie N, et al. miR-27a-3p is a negative regulator of lung fibrosis by targeting myofibroblast differentiation[J]. Am J Respir Cell Mol Biol, 2016, 54(6):843- 52.
[8] Li J M, Zhou J, Xu Z, et al. MicroRNA-27a-3p inhibits cell viability and migration through down-regulating DUSP16 in hepatocellular carcinoma[J]. J Cell Biochem, 2018, 119(7):5143-52.
[9] Liu J, Li M, Liu X, et al. miR-27a-3p promotes the malignant phenotypes of osteosarcoma by targeting ten-eleven translocation 1[J]. Int J Oncol, 2018, 52(4): 1295- 304.
[10] Matsuzaki S,Darcha C. Involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms of fibrosis in endometriosis[J]. PLoS One,2013, 8(10):e76808.
[11] Wang W,Zhao L,Ma Q, et al. The role of the Wnt/β-catenin pathway in the effect of implant topography on MG63 differentiation[J]. Biomaterials,2012, 33(32):7993-8002.
[12] Aumiller V, Balsara N, Wilhelm J, et al. Wnt/beta-catenin signaling induces IL-1beta expression by alveolar epithelial cells in pulmonary fibrosis[J]. Am J Respir Cell Mol Biol, 2013, 49(1):96-104.
[13] K?nigshoff M, Balsara N, Pfaff E M, et al. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis[J].PLoS One, 2008, 3(5):e2142.
[14] Guo Y, Mishra A, Howland E, et al. Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation[J]. Blood, 2015, 126(19):2220-9.
[15] Zhao Y,Wang P,Meng J, et al. MicroRNA-27a-3p inhibits melanogenesis in mouse skin melanocytes by targeting Wnt3a[J]. Int J Mol Sci,2015, 16(5):10921-33.
[16] Kim T H, Kim S H, Seo J Y, et al. Blockade of the Wnt/β-catenin pathway attenuates bleomycin-induced pulmonary fibrosis[J].Tohoku J Exp Med, 2011, 223(1):45-54.
[2] 刘理静,钱红,尹辉明,等. IL-17单克隆抗体对肺纤维化大鼠的保护作用及部分机制研究[J].中国药理学通报, 2015, 31(11):1586-92. Liu L J, Qian H, Yin H M, et al. Protective effect of IL-17 monoclonal antibody on rats with pulmonary fibrosis and its partial mechanisms[J]. Chin Pharmacol Bull, 2015, 31(11):1586-92.
[3] Wang Y, Liu J, Chen J, et al. MiR-29 mediates TGFβ1-induced extracellular matrix synthesis through activation of Wnt/β-catenin pathway in human pulmonary fibroblasts[J]. Technol Health Care, 2015, 23(Suppl 1):S119-25.
[4] Tan J, Tong B D, Wu Y J, et al. MicroRNA-29 mediates TGFβ1-induced extracellular matrix synthesis by targeting Wnt/β-catenin pathway in human orbital fibroblasts[J]. Int J Clin Exp Pathol, 2014, 7(11):7571-7.
[5] 刘理静, 钱红. 上调 miR-21表达促进肺成纤维细胞增殖和胶原蛋白合成[J]. 细胞与分子免疫学杂志, 2015, 31(7):918-22. Liu L J, Qian H. Up-regulation of miR-21 promotes cell proliferation and collagen synthesis in pulmonary fibroblasts[J]. Chin J Cell Mol Immunol, 2015, 31(7):918-22.
[6] Liu B, Li R, Zhang J, et al. MicroRNA-708-3p as a potential therapeutic target via the ADAM17-GATA/STAT3 axis in idiopathic pulmonary fibrosis[J]. Exp Mol Med, 2018, 50(3):e465.
[7] Cui H, Banerjee S, Xie N, et al. miR-27a-3p is a negative regulator of lung fibrosis by targeting myofibroblast differentiation[J]. Am J Respir Cell Mol Biol, 2016, 54(6):843- 52.
[8] Li J M, Zhou J, Xu Z, et al. MicroRNA-27a-3p inhibits cell viability and migration through down-regulating DUSP16 in hepatocellular carcinoma[J]. J Cell Biochem, 2018, 119(7):5143-52.
[9] Liu J, Li M, Liu X, et al. miR-27a-3p promotes the malignant phenotypes of osteosarcoma by targeting ten-eleven translocation 1[J]. Int J Oncol, 2018, 52(4): 1295- 304.
[10] Matsuzaki S,Darcha C. Involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms of fibrosis in endometriosis[J]. PLoS One,2013, 8(10):e76808.
[11] Wang W,Zhao L,Ma Q, et al. The role of the Wnt/β-catenin pathway in the effect of implant topography on MG63 differentiation[J]. Biomaterials,2012, 33(32):7993-8002.
[12] Aumiller V, Balsara N, Wilhelm J, et al. Wnt/beta-catenin signaling induces IL-1beta expression by alveolar epithelial cells in pulmonary fibrosis[J]. Am J Respir Cell Mol Biol, 2013, 49(1):96-104.
[13] K?nigshoff M, Balsara N, Pfaff E M, et al. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis[J].PLoS One, 2008, 3(5):e2142.
[14] Guo Y, Mishra A, Howland E, et al. Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation[J]. Blood, 2015, 126(19):2220-9.
[15] Zhao Y,Wang P,Meng J, et al. MicroRNA-27a-3p inhibits melanogenesis in mouse skin melanocytes by targeting Wnt3a[J]. Int J Mol Sci,2015, 16(5):10921-33.
[16] Kim T H, Kim S H, Seo J Y, et al. Blockade of the Wnt/β-catenin pathway attenuates bleomycin-induced pulmonary fibrosis[J].Tohoku J Exp Med, 2011, 223(1):45-54.
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