固肺消积饮上调外泌体microRNA-186-5p表达抑制肺癌A549细胞迁移和侵袭

Gufei Xiaoji Decoction inhibits lung cancer A549 cell migration and invasion by up-regulating exosomal microRNA-186-5p expression

目的:研究外泌体途径在固肺消积饮抑制肺癌A549细胞迁移和侵袭中的作用,及microRNA-186-5p(miR-186-5p)在其中的作用机制。方法:制备固肺消积饮含药血清和无药血清,分别诱导A549细胞、转染miR-186-5p NC的A549细胞、转染miR-186-5p inhibitor的A549细胞,获得4种外泌体,即NC-Exo、XJD-Exo、XJD-miR-186-5p NC-Exo、XJD-miR-186-5p inhibitor-Exo。透射电镜观察外泌体形态;Western blot检测CD63、TSG101和Calnexin表达;荧光标记法检测A549细胞摄取外泌体情况;RT-qPCR检测外泌体中miR-186-5p表达。实验分为:A组(A549细胞+PBS)、B组(A549细胞+NC-Exo)、C组(A549细胞+XJD-Exo)、D组(A549细胞+XJD-miR-186-5p NC-Exo)、E组(A549细胞+XJD-miR-186-5p inhibitor-Exo)。Transwell检测各组细胞迁移和侵袭;Western blot检测上皮-间质转化相关蛋白表达。结果:外泌体呈典型囊泡状,高表达表面标志物CD63和TSG101,低表达Calnexin,荧光标记的外泌体可被A549细胞大量摄取至胞浆内。NC-Exo、XJD-miR-186-5p inhibitor-Exo细胞miR-186-5p表达低于XJD-Exo、XJD-miR-186-5p NC-Exo细胞(P<0.05)。A、C、D组迁移、侵袭能力低于B、E组(P<0.05)。A、C、D组E-cadherin表达高于B、E组,N-cadherin、vimentin表达低于B、E组(P<0.05)。结论:固肺消积饮可通过上调外泌体miR-186-5p表达抑制肺癌A549细胞迁移和侵袭。

Objective:To investigate role of exosome pathway in inhibiting migration and invasion of lung cancer A549 cells by Gufei Xiaoji Decoction and mechanism of microRNA-186-5p(miR-186-5p).Methods:Gufei Xiaoji Decoction-containing serum and drug-free serum were prepared and used to stimulate A549 cells,A549 cells transfected with miR-186-5p NC,and A549 cells transfected with miR-186-5p inhibitor.Four exosomes,namely NC-Exo,XJD-Exo,XJD-miR-186-5p NC-Exo,XJD-miR-186-5p inhibitor-Exo,were obtained.Morphology of exosomes was observed by transmission electron microscope.Expressions of CD63,TSG101 and Calnexin were detected by Western blot.Uptake of exosomes by A549 cells was detected by fluorescent labeling.Expression of miR-186-5p in exosomes was detected by RT-qPCR.Experimental groups were:Group A(A549 cells+PBS),Group B(A549 cells+NC-Exo),Group C(A549 cells+XJD-Exo),Group D(A549 cells+XJD-miR-186-5p NC-Exo),Group E(A549 cells+XJDmiR-186-5p inhibitor-Exo).Migration and invasion of cells in each group were detected by Transwell.Expressions of epithelial-mesenchymal transition-related proteins were detected by Western blot.Results:Exosomes had a typical vesicle-like structure,with high expression of surface markers CD63 and TSG101,low expression of Calnexin.Fluorescently labeled exosomes could be taken up into cytoplasm by A549 cells.Expressions of miR-186-5p in NC-Exo and XJD-miR-186-5p inhibitor-Exo cells were lower than those of XJD-Exo and XJD-miR-186-5p NC-Exo cells(P<0.05).Migration and invasion abilities of groups A,C and D were lower than those of groups B and E(P<0.05).Expressions of E-cadherin in groups A,C and D were higher than those in groups B and E,and expressions of N-cadherin and vimentin were lower than those in groups B and E(P<0.05).Conclusion:Gufei Xiaoji Decoction can inhibit lung cancer A549 cell migration and invasion by up-regulating expression of miR-186-5p in exosomes.