脯氨酰顺反异构酶1调控低氧诱导内皮间充质转化的作用及机制

The role and mechanism of peptidyl-prolyl cis/trans isomerase never in mitosis A-interacting 1 in regulating hypoxia-induced endothelial-to-mesenchymal transition

目的探讨低氧条件下肺动脉内皮细胞(PAECs)中脯氨酰顺反异构酶(Pin1)的表达及其调控内皮间充质转化(EndMT)的作用及机制。方法利用低氧(1%O_(2))24 h建立低氧诱导的EndMT细胞模型。将PAECs分为4组,常氧组(Nor)、常氧+Pin1慢病毒敲低组(Nor+Pin1-shRNA)、低氧组(Hyp)、低氧+Pin1慢病毒敲低组(Hyp+Pin1-shRNA),分别培养24 h。应用5-溴-2-脱氧尿嘧啶(EdU)方法检测各组细胞增殖,细胞迁移实验(Transwell)检测细胞迁移。蛋白印迹法(Western blot)检测Pin1、缺氧诱导因子-1α(HIF-1α)的表达,并检测EndMT相关标志物α-平滑肌肌动蛋白(α-SMA)、血管内皮钙黏蛋白(VE-cadherin)、锌指转录因子(Snail)以及转化生长因子-β1(TGF-β1)相关信号通路的表达。多组间均数比较采用单因素方差分析。结果Nor+Pin1-shRNA组Pin1的蛋白相对表达量(0.292±0.061)明显低于Nor组(0.798±0.054),Hyp+Pin1-shRNA组Pin1的蛋白相对表达量(0.475±0.039)明显低于Hyp组(1.403±0.076),差异有统计学意义(F=271.983,P<0.01);Hyp组(1.135±0.086)和Hyp+Pin1-shRNA组(1.043±0.054)HIF-1α的蛋白相对表达量明显高于Nor组(0.735±0.042),差异有统计学意义(F=60.954,P<0.01);Hyp组(20.500±1.871)EdU阳性细胞数明显高于Nor组(7.167±1.169),Hyp+Pin1-shRNA组(9.327±1.633)EdU阳性细胞数明显低于Hyp组,差异有统计学意义(F=128.416,P均<0.01);Hyp组(464.500±10.599)细胞数明显高于Nor组(179.250±11.955),Hyp+Pin1-shRNA组(320.000±17.607)细胞数明显低于Hyp组,差异有统计学意义(F=501.244,P<0.01);Hyp组(1.205±0.061)α-SMA的蛋白相对表达量明显高于Nor组(0.725±0.089),Hyp+Pin1-shRNA组α-SMA的蛋白相对表达量(0.870±0.058)明显低于Hyp组,差异有统计学意义(F=50.820,P<0.01);Hyp组(0.547±0.066)VE-cadherin的蛋白相对表达量明显低于Nor组(0.975±0.079),Hyp+Pin1-shRNA组(0.797±0.051)VE-cadherin的蛋白相对表达量明显高于Hyp组,差异有统计学意义(F=27.934,P<0.01);Hyp组Snail(0.955±0.063)、TGF-β1(1.042±0.087)、p-Smad2(1.180±0.062)的蛋白相对表达量明显高于Nor组(0.510±0.036、0.647±0.053、0.807±0.053),Hyp+Pin1-shRNA组Snail(0.492±0.061)、TGF-β1(0.792±0.051)、p-Smad2(0.910±0.036)的蛋白相对表达量明显低于Hyp组,差异有统计学意义(F=82.842、38.131、48.617,P<0.01)。结论低氧条件下,PAECs中Pin1表达升高,并发生EndMT,抑制Pin1可减弱EndMT,其机制是Pin1通过影响TGF-β1-Smad信号通路,促进Snail等转录因子的表达。

Objective To investigate the expression of peptidyl-prolyl cis/trans isomerase never in mitosis A-interacting 1(Pin1)in pulmonary arterial endothelial cells(PAECs)under hypoxic conditions and its role in regulating endothelial-to-mesenchymal transition(EndMT).Methods A hypoxia-induced(1%O_(2))EndMT cell model was used to test effect of Pin1.PAECs were divided into 4 groups:normoxia group(Nor),normoxia with Pin1 knockdown group(Nor+Pin1-shRNA),hypoxia group(Hyp),and hypoxia with Pin1 knockdown group(Hyp+Pin1-shRNA).Each group was cultured for 24 h.Cell proliferation and migration were assessed separately using the 5-bromo-2’-deoxyuridine(EdU)assay and Transwell migration assay,respectively.Western blotting was conducted to assess the expression levels of Pin1 and hypoxia-inducible factor-1 alpha(HIF-1α).Additionally,the expression of EndMT-related markers includingα-smooth muscle actin(α-SMA),vascular endothelial cadherin(VE-cadherin),Snail,and the transforming growth factor-beta 1(TGF-β1)-related signaling were also detected.Multiple group mean comparisons were conducted using one-way analysis of variance.Results The relative protein expression of Pin1 in the Nor+Pin1-shRNA group(0.292±0.061)was significantly lower than Nor group(0.798±0.054).Similarly,the relative protein expression of Pin1 in the Hyp+Pin1-shRNA group(0.475±0.039)was significantly lower than in the Hyp group(1.403±0.076).(F=271.983,P<0.01).The relative protein expression of HIF-1αwas significantly higher in both the Hyp group(1.135±0.086)and the Hyp+Pin1-shRNA group(1.043±0.054)than in the Nor group(0.735±0.042,F=60.954,P<0.01).The number of EdU-positive cells in the Hyp group(20.500±1.871)was significantly greater than in the Nor group(7.167±1.169).In contrast,the Hyp+Pin1-shRNA group(9.327±1.633)exhibited a significantly less number of EdU-positive cells than the Hyp group(F=128.416,P<0.01).The cell count in the Hyp group(464.500±10.599)was significantly greater than in the Nor group(179.250±11.955).Conversely,the Hyp+Pin1-shRNA group(320.000±17.607)exhibited a significantly less cell count than the Hyp group(F=501.244,P<0.01).The relative protein expression ofα-SMA in the Hyp group(1.205±0.061)was significantly higher than in the Nor group(0.725±0.089),and that in the Hyp+Pin1-shRNA group(0.870±0.058)was significantly lower than in the Hyp group(F=50.820,P<0.01).The relative protein expression of VE-cadherin in the Hyp group(0.547±0.066)was significantly lower than in the Nor group(0.975±0.079),and that in the Hyp+Pin1-shRNA group(0.797±0.051)was significantly higher than in the Hyp group(F=27.934,P<0.01).The relative protein expression of Snail(0.955±0.063),TGF-β1(1.042±0.087),and p-Smad2(1.180±0.062)in the Hyp group was significantly higher than in the Nor group(0.510±0.036,0.647±0.053,0.807±0.053).In the Hyp+Pin1-shRNA group,the expression of Snail(0.492±0.061),TGF-β1(0.792±0.051),and p-Smad2(0.910±0.036)was significantly lower than in the Hyp group(F=82.842,38.131,48.617,P<0.01).Conclusion Under hypoxic conditions,the expression of Pin1 in PAECs is upregulated,accompanied by the occurrence of EndMT.Inhibiting Pin1 attenuates EndoMT by activating the TGF-β1-Smad2 signaling pathway and promoting the expression of the transcription factor Snail.