肝癌细胞外泌体诱导巨噬细胞M2极化后促进肝癌细胞迁移及侵袭

M2 polarized macrophages induced by exosomes of hepatocellular carcinoma cells prompt migration and invasion of hepatocellular carcinoma cells

目的:探讨外泌体在肝细胞癌(HCC)细胞与巨噬细胞间是否存在活性物质交通作用,分析肿瘤微环境中肿瘤相关巨噬细胞(TAMs)与HCC细胞转移的关系及机制。方法:采用超速离心法从Huh-7和RAW264.7细胞的培养基中提取外泌体,Western blot和透射电镜进行外泌体鉴定;激光共聚焦显微镜对外泌体与受体细胞结合内化进行追踪;qRT-PCR及ELISA检测巨噬细胞表型变化;miRNA模拟物进行功能性实验验证其作用;细胞划痕及Transwell试验检测TAMs对HCC细胞迁移及侵袭能力的影响。结果:HCC细胞外泌体可被巨噬细胞内吞;肝癌细胞外泌体及miR-151模拟物处理的巨噬细胞M2型标志物CD206、Arg-1 mRNA表达及IL-10、TGF-β蛋白表达显著高于对照组(P<0.05);TAMs外泌体处理组HCC细胞迁移及侵袭能力高于其他组(P<0.05)。结论:HCC细胞来源外泌体miR-151可诱导巨噬细胞极化为TAMs,TAMs对肝癌细胞迁移及侵袭有促进作用。

Objective:To investigate whether there are communications of exosomes between hepatocellular carcinoma(HCC)cells and macrophages,and relationship between tumor-associated macrophages(TAMs)in tumor microenvironment and HCC metastasis and its mechanism.Methods:Exosomes of Huh-7 cells and RAW264.7 cells were extracted from culture medium by ultracentrifugation,and identified by Western blot and transmission electron microscopy.Laser confocal microscopy to track binding and internalization of exosomes to recipient cells.Phenotypic changes of macrophages were detected by qRT-PCR and ELISA.Functional experiments of miRNA mimics were performed to verify their effects.Effect of TAMs on migration and invasion of HCC cells was detected by wound healing and Transwell assay.Results:Exosomes of HCC cells could be endocytosis by macrophages.mRNA expressions of M2 type markers CD206 and Arg-1 and protein expressions of IL-10 and TGF-βin macrophages treated with HCC exosomes and miR-151 mimics were significantly higher than those in control group(P<0.05).Migration and invasion abilities of HCC cells in TAMS exosome treatment group were higher than those in other groups(P<0.05).Conclusion:Exosomal miR-151 derived from HCC cells may induce macrophages polarizing into TAMs,and exosomes secreted by TAMs may promote migration and invasion of liver cancer cells.