摘要
目的探讨色素上皮衍生因子(PEDF)对转化生长因子-β1(TGF-β1)诱导的肺癌A549细胞上皮-间充质转化(EMT)进程的影响及其分子机制。方法选取肺癌A459细胞(购自中科院上海细胞生物研究所)为研究对象,使用TGF-β1建立EMT模型,分为正常组、TGF-β1处理组(TGF-β1,10 ng/ml)、PEDF处理组(TGF-β1,10 ng/ml;PEDF,20 nmol/L)和氯化锂(GSK-3β抑制剂,LiCl)处理组(TGF-β1,10 ng/ml;PEDF,20 nmol/L;LiCl,20 mmol/L)。光镜下观察各组A549细胞的形态变化;划痕实验观察细胞的迁移能力;免疫荧光法和免疫印迹法观察和检测各组钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的表达和分布。两组间比较采用t检验。结果TGF-β1促使A549细胞呈狭长纺锤形变化,显著增加其迁移能力;PEDF部分逆转A549形态改变和迁移能力的增加;LiCl消除了PEDF的逆转作用。免疫荧光和免疫印迹结果显示,TGF-β1组α-SMA(0.973±0.081)、p-GSK-3β^(Ser9)(0.875±0.065)和snail(0.562±0.049)高于正常组α-SMA(0.594±0.041)、p-GSK-3β^(Ser9)(0.267±0.043)、snail(0.222±0.041,t=7.283、13.886、11.634,P值均<0.05),TGF-β1组E-cadherin(0.442±0.066)低于正常组E-cadherin(0.892±0.052,t=10.378,P<0.05);PEDF组α-SMA(0.712±0.063)、p-GSK-3β^(Ser9)(0.566±0.067)和snail(0.354±0.056)低于TGF-β1组α-SMA(0.973±0.081)、p-GSK-3β^(Ser9)(0.875±0.065)和snail(0.562±0.049,t=4.383、6.256、5.927,P值均<0.05),PEDF组E-cadherin(0.785±0.071)高于TGF-β1组E-cadherin(0.442±0.066,t=6.845,P<0.05);LiCl组α-SMA(1.021±0.083)、p-GSK-3β^(Ser9)(0.750±0.050)和snail(0.687±0.032)高于PEDF组α-SMA(0.712±0.063)、p-GSK-3β^(Ser9)(0.566±0.067)和snail(0.354±0.056,t=4.868、4.276、9.667,P值均<0.05),LiCl组E-cadherin(0.385±0.056)低于PEDF组E-cadherin(0.785±0.071,t=8.633,P<0.05)。结论PEDF通过调节p-GSK-3β^(Ser9)/snail信号通路抑制TGF-β1诱导的A549细胞EMT进程。
Objective To investigate the effect of pigment epithelium-derived factor(PEDF)on the process of epithelial mesenchymal transition(EMT)in lung cancer A549 cells induced by transforming growth factor-β1(TGF-β1)and its molecular mechanism.Methods A549 cells were incubated with TGF-β1 to establish EMT model.Normal group,TGF-β1(10 ng/ml)group,PEDF group(TGF-β1,10 ng/ml;PEDF,20 nmol/L)and lithium chloride(GSK-3βinhibitor)group(TGF-β1,10 ng/ml;PEDF,20 nmol/L;LiCl,20 mmol/L)were set up.The morphological changes of A549 cells were observed under light microscope.Cell scratch test was used to detect cell migration ability.The expression and distribution of E-cadherin andα-smooth muscle actin(α-SMA)were detected by immunofluorescence and Western blotting.Data were analyzed using the independent-samples t test for two group comparisons.Results TGF-β1 promoted the elongated,spindle-shaped changes of A549 cells and significantly increased the migration ability.PEDF partially reversed the morphological changes and the increase of migration ability of A549 cells,and LiCl eliminated the reversal effect of PEDF.Western blotting results showed thatα-SMA(0.973±0.081),p-GSK-3β^(Ser9)(0.875±0.065)and snail(0.562±0.049)in the TGF-β1 group were significantly higher than those in the normal group[α-SMA:(0.594±0.041);p-GSK-3β^(Ser9):(0.267±0.043);snail:(0.222±0.041);t=7.283,13.886,11.634,P<0.05].E-cadherin(0.442±0.066)in TGF-β1 group was significantly lower than that in normal group(0.892±0.052,t=10.378,P<0.05).In the PEDF group,α-SMA(0.712±0.063),p-GSK-3β^(Ser9)(0.566±0.067)and snail(0.354±0.056)were significantly lower than in the TGF-β1 group[α-SMA:(0.973±0.081);p-GSK-3β^(Ser9):(0.875±0.065);snail:(0.562±0.049),t=4.383,6.256,5.927,P<0.05].In PEDF group,E-cadherin(0.785±0.071)was significantly higher than in TGF-β1 group[(0.442±0.066),t=6.845,P<0.05].α-SMA(1.021±0.083),p-GSK-3β^(Ser9)(0.750±0.050)and snail(0.687±0.032)in LiCl group were significantly higher than tin he PEDF group[α-SMA:(0.712�
作者
贾才力
秦西淳
刘修成
张昊
Jia Caili;Qin Xichun;Liu Xiucheng;Zhang Hao(Department of Thoracic Surgery,Affiliated Hospital of Xuzhou Medical University,Xuzhou 221002,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第8期1409-1412,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81400227)
江苏省社会发展重点研发计划(BE2019643)
江苏省自然科学基金(BK20171178)
江苏省卫生与健康委员会面上项目(H2017083)
江苏省科教强卫青年人才项目(QNRC2016778)。
