HAX-1通过调控MDM2/SMAD3通路对TGF-β1诱导的特发性肺间质纤维化的作用机制

Mechanism of HAX-1 on TGF-β1-induced Idiopathic Pulmonary Fibrosis by Regulating MDM2/SMAD3 Pathway

目的探讨造血细胞特异性蛋白1相关蛋白X-1(HAX-1)对转化生长因子-β1(TGF-β1)诱导的特发性肺间质纤维化的作用及机制。方法将A549细胞分为对照组(无处理)、TGF-β1组(用5μg·L^(-1)的TGF-β1诱导48 h)、NC si组(用阴性对照siRNA转染细胞,再用5μg·L^(-1)的TGF-β1诱导48 h)、HAX-1 si组(用HAX-1 siRNA转染细胞,再用5μg·L^(-1)的TGF-β1诱导48 h)、HAX-1 si+pcDNA3.1组(用HAX-1 siRNA转染细胞,再用pcDNA3.1空质粒转染细胞,再用5μg·L^(-1)的TGF-β1诱导48 h)、HAX-1 si+MDM2组(用HAX-1 siRNA转染细胞,再用MDM2过表达质粒pcDNA3.1-MDM2转染细胞,再用5μg·L^(-1)的TGF-β1诱导48 h)、HAX-1 si+MDM2+DMSO组(用HAX-1 siRNA转染细胞,再用MDM2过表达质粒pcDNA3.1-MDM2转染细胞,向细胞培养液中加入溶剂DMSO处理细胞30 min,再用5μg·L^(-1)的TGF-β1诱导48 h)、HAX-1 si+MDM2+SIS3组(用HAX-1 siRNA转染细胞,再用MDM2过表达质粒pcDNA3.1-MDM2转染细胞,用终浓度为1μmol·L^(-1)溶于DMSO的SIS3处理细胞30 min,再用5μg·L^(-1)的TGF-β1诱导48 h)。相应处理结束后,采用倒置相差显微镜观察细胞形态变化;Western blot检测HAX-1、鼠双微体基因2(MDM2)、SMAD3、p-SMAD3、E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原α1链(COL1A1)蛋白表达水平。结果TGF-β1诱导细胞后,细胞由鹅卵石形或多角形转变为梭形、纺锤形,E-cadherin蛋白表达降低(P<0.05),HAX-1、α-SMA和COL1A1蛋白表达升高(P<0.05)。干扰HAX-1可提高E-cadherin蛋白表达(P<0.05),抑制MDM2、p-SMAD3、α-SMA和COL1A1蛋白表达(P<0.05),并且细胞形态又转变为鹅卵石形或多角形。MDM2过表达抵消HAX-1干扰对p-SMAD3、E-cadherin、α-SMA和COL1A1蛋白表达的影响(P<0.05)。SMAD3抑制剂SIS3处理后细胞形态又转变为鹅卵石形或多角形,E-cadherin蛋白表达升高(P<0.05),α-SMA和COL1A1蛋白表达下降(P<0.05)。结论HAX-1在TGF-β1诱导的A549细胞中高表达,HAX-1敲低可通过MDM2/SMAD3通路抑制TGF-β1诱导的肺泡细胞纤维化过程。

Objective To explore the effect and underlying mechanism of hematopoietic cell specific protein 1 associated protein X-1(HAX-1)on transforming growth factor-β1(TGF-β1)-induced idiopathic pulmonary fibrosis.Methods A549 cells were divided into following groups:control group(no treatment),TGF-β1 group(cells were induced with 5μg·L^(-1) TGF-β1 for 48 hours),NC si group(cells were transfected with negative control siRNA,and were then induced with 5μg·L^(-1) TGF-β1 for 48 hours),HAX-1 si group(cells were transfected with HAX-1 siRNA,and were then induced with 5μg·L^(-1) TGF-β1 for 48 hours),HAX-1 si+pcDNA3.1 group(cells were transfected with HAX-1 siRNA and pcDNA3.1 empty plasmids,and were then induced with 5μg·L^(-1) TGF-β1 for 48 hours),HAX-1 si+MDM2 group(cells were transfected with HAX-1 siRNA and MDM2 overexpression plasmids,and were then induced with 5μg·L^(-1) TGF-β1 for 48 hours),HAX-1 si+MDM2+DMSO group(cells were transfected with HAX-1 siRNA and MDM2 overexpression plasmids,then cells were treated with DMSO for 30 minutes,and then cells were induced with 5μg·L^(-1) TGF-β1 for 48 hours)and HAX-1 si+MDM2+SIS3 group(cells were transfected with HAX-1 siRNA and MDM2 overexpression plasmids,then cells were treated with 1μmol·L^(-1) SIS3 for 30 minutes,and then cells were induced with 5μg·L^(-1) TGF-β1 for 48 hours).After the corresponding treatment,the inverted phase-contrast microscope was used to observe the changes in cell morphology.Western blot was used to detect the protein expression of HAX-1,murine double minute 2(MDM2),SMAD3,p-SMAD3,E-cadherin,α-smooth muscle actin(α-SMA)and collagen typeⅠalpha 1 chain(COL1A1).Results After TGF-β1 induction,the cells changed from cobblestone or polygonal to fusiform and spindle-shaped.E-cadherin protein expression was reduced(P<0.05),and HAX-1,α-SMA and COL1A1 protein expression were improved(P<0.05).HAX-1 knockdown increased the protein expression of E-cadherin(P<0.05),and inhibited the protein expression of MDM2,p-SMAD3,α-SMA and COL1A1(P<0.05),and the cell morphs recovered into pebble or polygon.MDM2 overexpression offsets the effect of HAX-1 knockdown on p-SMAD3,E-cadherin,α-SMA and COL1A1 protein expressions(P<0.05).After induced by the SMAD3 inhibitor,SIS3,the cell morphology changed to a cobblestone or polygonal shape,and the expression of E-cadherin protein increased(P<0.05),the expression ofα-SMA and COL1A1 protein decreased(P<0.05).Conclusion HAX-1 was highly expressed in the TGF-β1 induced A549 cells.HAX-1 knockdown could reduce the TGF-β1 induced pulmonary fibrosis by regulating the MDM2/SMAD3 pathway.