Molecular mechanisms of Yes-associated protein in regulating the proliferation of pulmonary arterial smooth muscle cells
柯蕊 1和平 1张伟 1史文花 1张永红 1刘原 2王秋红
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作者信息
1. 西安交通大学第二附属医院呼吸与危重症医学科，西安 710004
2. 西安交通大学第二附属医院健康体检部，西安 710004
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摘要
目的 研究Yes相关蛋白(YAP)调控肺动脉平滑肌细胞(PASMCs)增殖的分子机制。 方法 本研究为实验研究。选取健康SD大鼠，体质量70～80 g。培养大鼠PASMCs，根据是否给予S1P刺激将PASMCs分为S1P组和对照组，蛋白质印迹法检测细胞磷酸化YAP(p-YAP)和β-catenin、CyclinD1蛋白水平。按照转染control干扰小RNA(siRNA)或YAP siRNA后再给予S1P刺激的情况不同，将PASMCs分为对照组、S1P组、si＋S1P组、si-YAP＋S1P组，蛋白质印迹法检测各组细胞中β-catenin和CyclinD1的蛋白水平。按照转染control siRNA或β-catenin siRNA后再给予S1P刺激的情况不同，将PASMCs分为对照组、S1P组、si＋S1P组、si-β-catenin＋S1P组，蛋白质印迹法检测各组细胞中CyclinD1的蛋白水平。按照转染control siRNA或YAP siRNA或β-catenin siRNA后再给予S1P刺激的情况不同，将PASMCs分为对照组、S1P组、si＋S1P组、si-YAP＋S1P组和si-β-catenin＋S1P组，BrdU掺入法检测各组细胞增殖情况。 结果 S1P组p-YAP水平低于对照组[(0.61±0.09)比(1.00±0.11)，P＝0.009]，β-catenin和CyclinD1蛋白水平高于对照组[(1.98±0.14)比(1.00±0.10)，(1.94±0.15)比(1.00±0.08)，均P＝0.001]。si-YAP＋S1P组β-catenin、CyclinD1水平均低于S1P组[(1.15±0.09)比(1.95±0.12)，(1.18±0.16)比(1.96±0.05)，均P<0.01]。si-β-catenin＋S1P组CyclinD1水平低于S1P组[(1.18±0.24)比(1.95±0.24)，P<0.01]。S1P组细胞增殖率高于对照组[(169.69±12.85)%比(100.00±12.52)%，P<0.01]，si-YAP＋S1P组、si-β-catenin＋S1P组细胞增殖率均低于S1P组[(126.33±14.56)%比(169.69±12.85)%，(128.10±15.25)%比(169.69±12.85)%，均P<0.01]。 结论 YAP/β-catenin/CyclinD1信号通路可以调控PASMCs增殖。 Objective To examine the molecular mechanisms of Yes-associated protein (YAP) in regulating the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Methods It was an experimental study.PASMCs were separated from healthy Sprague-Dawley (SD) rats weighing 70-80 g. Primary PASMCs were cultured and stimulated with S1P or blank control, followed by the detection of the expressions of phosphorylated YAP (p-YAP), β-catenin and CyclinD1 by Western blot.Then, PASMCs were induced with blank control, S1P, S1P+ transfection of si-NC, and S1P+ transfection of si-YAP, followed by the detection of the protein expressions of β-catenin and CyclinD1 by Western blot and assessment of cell proliferation by BrdU assay.PASMCs were further induced with blank control, S1P, S1P+ transfection of si-NC, and S1P+ transfection of si-β-catenin, followed by the detection of the protein expression of CyclinD1 by Western blot and assessment of cell proliferation by BrdU assay. Results The expression of p-YAP ([0.61±0.09] vs [1.00±0.11], P=0.009) was significantly lower in S1P-induced PASMCs than that of blank control, while protein expressions of β-catenin ([1.98±0.14] vs [1.00±0.10], P=0.001) and CyclinD1 ([1.94±0.15] vs [1.00±0.08], P=0.001) were significantly higher.Protein expressions of β-catenin ([1.15±0.09] vs [1.95±0.12], P<0.01) and CyclinD1 ([1.18±0.16]vs [1.96±0.05], P<0.01) were significantly lower in PASMCs induced with S1P+ transfection of si-YAP than those induced with S1P.Protein expression of CyclinD1 ([1.18±0.24]vs [1.95±0.24], P<0.01) was significantly lower in PASMCs induced with S1P+ transfection of si-β-catenin than those induced with S1P.The proliferative rate was significantly higher in PASMCs induced with S1P ([169.69±12.85]%vs [100.00±12.52]%, P<0.01) than that of blank control.The proliferative rate was significantly lower in PASMCs induced with S1P+ transfection of si-YAP ([126.33±14.56]%vs [169.69±12.85]%, P<0.01) and S1P+ transfection of si-β-catenin ([128.10±15.25]%vs [169.69±12.85]%, P<0.01) than that induced with S1P. Conclusions The YAP/β-catenin/CyclinD1 signaling pathway promotes proliferation of PASMCs.
Abstract
Objective To examine the molecular mechanisms of Yes-associated protein (YAP) in regulating the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Methods It was an experimental study.PASMCs were separated from healthy Sprague-Dawley (SD) rats weighing 70-80 g. Primary PASMCs were cultured and stimulated with S1P or blank control, followed by the detection of the expressions of phosphorylated YAP (p-YAP), β-catenin and CyclinD1 by Western blot.Then, PASMCs were induced with blank control, S1P, S1P+ transfection of si-NC, and S1P+ transfection of si-YAP, followed by the detection of the protein expressions of β-catenin and CyclinD1 by Western blot and assessment of cell proliferation by BrdU assay.PASMCs were further induced with blank control, S1P, S1P+ transfection of si-NC, and S1P+ transfection of si-β-catenin, followed by the detection of the protein expression of CyclinD1 by Western blot and assessment of cell proliferation by BrdU assay. Results The expression of p-YAP ([0.61±0.09] vs [1.00±0.11], P=0.009) was significantly lower in S1P-induced PASMCs than that of blank control, while protein expressions of β-catenin ([1.98±0.14] vs [1.00±0.10], P=0.001) and CyclinD1 ([1.94±0.15] vs [1.00±0.08], P=0.001) were significantly higher.Protein expressions of β-catenin ([1.15±0.09] vs [1.95±0.12], P<0.01) and CyclinD1 ([1.18±0.16]vs [1.96±0.05], P<0.01) were significantly lower in PASMCs induced with S1P+ transfection of si-YAP than those induced with S1P.Protein expression of CyclinD1 ([1.18±0.24]vs [1.95±0.24], P<0.01) was significantly lower in PASMCs induced with S1P+ transfection of si-β-catenin than those induced with S1P.The proliferative rate was significantly higher in PASMCs induced with S1P ([169.69±12.85]%vs [100.00±12.52]%, P<0.01) than that of blank control.The proliferative rate was significantly lower in PASMCs induced with S1P+ transfection of si-YAP ([126.33±14.56]%vs [169.69±12.85]%, P<0.01) and S1P+ transfection of si-β-catenin ([128.10±15.25]%vs [169.69±12.85]%, P<0.01) than that induced with S1P. Conclusions The YAP/β-catenin/CyclinD1 signaling pathway promotes proliferation of PASMCs.
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