内质网通道蛋白Sec61在PM2.5诱导支气管上皮细胞BEAS-2B高表达MUC5AC中的作用

Role of endoplasmic reticulum channel protein Sec61 in PM2.5-induced overexpression of MUC5AC in bronchial epithelial cells BEAS-2B

目的:探讨内质网通道蛋白Sec61在PM2.5诱导支气管上皮细胞BEAS-2B高表达黏蛋白5AC(MUC5AC)中的作用。方法:使用不同浓度的PM2.5(0、25、50、100、200μg/ml)刺激BEAS-2B不同时间(0、6、12、24、48 h),蛋白质免疫印迹和免疫荧光染色检测细胞中Sec61的表达。实验分为4组,即对照组、PM2.5组、siRNA-Control组、siRNA-Sec61组,除对照组外其余三组均进行PM2.5刺激,siRNA-Control组和siRNA-Sec61组分别使用转染对照或Sec61干扰序列的细胞。荧光定量聚合酶链式反应和Western blot检测细胞中Sec61、MUC5AC、GRP78的表达,免疫荧光染色检测细胞中MUC5AC和游离钙离子的表达。结果:随着PM2.5刺激浓度增加和刺激时间延长,Sec61的表达量逐渐升高,100μg/ml刺激48 h时,Sec61的表达量最高。与PM2.5组和siRNA-Control组相比,siRNA-Sec61组细胞中Sec61 mRNA和蛋白质的表达量降低(P<0.05);MUC5AC mRNA和蛋白质的表达量降低(P<0.05);GRP78 mRNA和蛋白质的表达量降低(P<0.05);细胞中游离钙离子的荧光强度明显降低。结论:PM2.5可以诱导Sec61高表达,呈浓度依赖性和时间依赖性。使用小分子RNA沉默Sec61表达可以显著抑制MUC5AC高表达,可能与调节内质网应激和钙离子稳态有关。

Objective:To explore the role of endoplasmic reticulum channel protein Sec61 in PM2.5-induced overexpression of MUC5AC in bronchial epithelial cells BEAS-2B.Methods:Different concentrations of PM2.5(0,25,50,100,200μg/ml)were used to stimulate BEAS-2B cells at different times(0,6,12,24,48 h).The expression of Sec61 in BEAS-2B cells was detected by Western blot and immunofluorescence staining.The experiment was divided into four groups:control group,PM2.5 group,siRNA-Control group and siRNA-Sec61 group.Except the control group,the cells in the other three groups were stimulated by PM2.5.The cells in the siRNA-Control group and siRNA-Sec61 group were transfected by the control or Sec61 interference sequence respectively.The expressions of Sec61,MUC5AC and GRP78 were detected by fluorescence quantitative polymerase chain reaction and Western blot.The expressions of MUC5AC and free calcium ions were detected by immunofluorescence staining.Results:With the increase of PM2.5 stimulation concentration and the prolongation of stimulation time,the expression of Sec61 increased gradually.The highest expression of Sec61 was observed at 48 h with the concentration of 100μg/ml.Compared with PM2.5 group and siRNA-Control group,the mRNA and protein expression of Sec61 decreased in siRNA-Sec61 group(P<0.05).The mRNA and protein expression of MUC5AC decreased(P<0.05).The mRNA and protein expression of GRP78 decreased(P<0.05)and the fluorescence intensity of free calcium ions decreased significantly in siRNA-Sec61 group.Conclusion:PM2.5 could induce Sec61 overexpression in a concentration-and time-dependent manner.Inhibition of Sec61 expression by small interference RNA significantly inhibited the overexpression of MUC5AC,which may be related to the regulation of endoplasmic reticulum stress and calcium homeostasis.