本研究旨在探究牛结核分枝杆菌减毒株卡介苗(Bacillus Calmette-Guérin,BCG)感染人单核巨噬细胞THP-1后,钙结合蛋白S100A4对细胞自噬的调控作用.以感染复数为10:1的BCG感染THP-1细胞不同时间,用Western blot检测S100A4和LC3 Ⅱ的表达,以确定最佳感染时间,并采用透射电镜观察自噬体数量变化.在BCG单独感染或与S100A4小干扰RNA共处理THP-1细胞12 h后,采用qRT-PCR检测S100A4及自噬相关因子 微管相关蛋白轻链 3Ⅱ(microtubule-associated protein light chain 3 Ⅱ,LC3 Ⅱ)、自噬相关蛋白 7(autoph-agy-related gene 7,Atg7)、Beclin-1 在 mRNA 水平上的表达,采用 Western blot 检测 S100A4、LC3Ⅱ、Atg7、Beclin-1在蛋白水平的表达.利用mRFP-GFP-LC3检测自噬流,激光共聚焦显微镜观察细胞中mRFP-LC3和mRFP-GFP-LC3点状聚集.结果显示:在BCG感染THP-1细胞不同时间后,S100A4和LC3 Ⅱ在蛋白表达水平随感染时间的延长先上升后下降,在12 h时表达最高(P<0.001),且自噬体数量明显增多.在mRNA水平上,与siNC组相比,siNC+BCG 感染组 S100A4、LC3 Ⅱ、Atg7、Beclin-i 的 mRNA 表达水平显著上调(P<0.05),当 BCG 和siS100A4 共处理后,S100A4、LC3 Ⅱ、Atg7、Beclin-1 在 mRNA 水平的表达显著(P<0.05)或极显著(P<0.01,P<0.001)下调;在蛋白水平上,与未感染组相比,S100A4、LC3 Ⅱ、Atg7、Beclin-1的蛋白表达量显著增多(P<0.05),BCG和siS100A4共处理后,S100A4、LC3Ⅱ、Atg7、Beclin-1的蛋白表达量均极显著减少(P<0.001);与未感染组相比,BCG组自噬体的数量极显著增多(P<0.01),siS100A4+BCG组与siNC+BCG组相比,自噬体的数量显著减少(P<0.05).S100A4对BCG感染巨噬细胞诱导的自噬具有调控作用,S100A4能够促进BCG诱导的THP-1细胞自噬.
The purpose of this study was to explore the regulatory effect of calcium-binding protein S100A4 on autophagy of human monocyte-macrophage THP-1 cells infected with Bacillus Calmette-Guérin(BCG).BCG infected THP-1 cells at different times(MOI=10).The expressions of S100A4 and LC3 proteins were detected by Western blot,and the number of autophagosomes was observed by transmission electron microscopy(TEM).After BCG infection alone or co-treatment with S100A4 small interfering RNA for 12 h,the mRNA expression of S100A4 and autophagy-related factors,LC3 Ⅱ(microtubule-associated protein light chain 3 Ⅱ),Atg7(autophagy-related gene 7),and Beclin1,were detected by qRT-PCR.The protein expressions of S100A4,LC3 Ⅱ,Atg7,and Beclin-1 were detected by Western blot.Autophagy flow was detected by mRFP-GFP-LC3,and spot aggregation of mRFP-LC3 and mRFP-GFP-LC3 was observed by laser confocal microscopy.After BCG was infected with THP-1 cells at different times,the protein expression levels of S100A4 and LC3 Ⅱ firstly increased and then decreased with the extension of infection time,and the expression levels were the highest at 12 h(P<0.001),and the number of autophagosomes increased significantly.mRNA levels of S100A4,LC3Ⅱ,Atg7,and Beclin-1 in the siNC+BCG infection group were significantly up-regulated compared with those in the siNC group(P<0.05),S100A4,LC3 Ⅱ,Atg7,and Beclin-1 were significantly expressed at mRNA level after BCG and siS100A4 treatment(P<0.05)or very significantly(P<0.01,P<0.001)down-regulated;Protein levels of S100A4,LC3 Ⅱ,Atg7,and Beclin-1 increased significantly compared with an uninfected group(P<0.05),the protein expression levels of S100A4,LC3 Ⅱ,Atg7,and Beclin-1 were significantly reduced after BCG and siS100A4 treatment(P<0.001);Compared with the uninfected group,the number of autophagosomes in BCG group was significantly increased(P<0.01),the number of autophagosomes in siS 100A4+BCG group was significantly reduced compared with siNC+BCG infection group(P<0.05).S100A4 has a regulatory effect on the autophagy of macrophages induced by BCG infection,and S100A4 can promote the BCG-induced autophagy of THP-1 cells.