低氧肺癌来源的外泌体微小RNA-1278对巨噬细胞M2极化的调节作用及其机制

Regulatory effect and mechanism of hypoxia-induced lung cancer cell-derived exosomal microRNA-1278 on M2-polarization of macrophages

目的探讨低氧肺癌细胞来源的外泌体微小RNA(miR)-1278对巨噬细胞M2极化的调节作用及其机制。方法收集正常条件和缺氧肺癌细胞的培养上清液,差速离心法分离外泌体,透射电子显微镜观察并计数,实时定量聚合酶链反应(PCR)检测miR-1278的表达水平。运用收集的外泌体(各10μg)处理CD14+单核细胞(主要是巨噬细胞),诱导细胞M2型分化,流式细胞术评估分化效果。miR-1278 mimic、miR-1278 inhibitor以及各自的阴性对照转染CD14+单核细胞,检测M2型巨噬细胞比例,酶联免疫吸附试验(ELISA)检测白细胞介素-10(IL-10)、趋化因子CC配体18(CCL-18)和血管内皮生长因子-A(VEGF-A)的分泌量。预测、筛选和验证miR-1278的靶基因,研究miR-1278对靶基因及其下游通路的调节作用。常氧和低氧CL1-5细胞外泌体分别与通路抑制剂WP1066或miR-1278 inhibitor处理CD14+单核细胞,检测M2型巨噬细胞比例,并研究巨噬细胞条件培养基对肺癌细胞迁移和内皮细胞血管形成的作用。采用Student′s t检验分析两组间差异,多组间数据差异采用方差分析进行比较。结果与常氧肺癌细胞比较,低氧肺癌细胞分泌的外泌体数量显著增加(0.8~1.5倍,P<0.05)。过表达和抑制miR-1278的水平分别促进常氧(12.06%比38.21%,P<0.05)和抑制低氧(30.55%比9.53%,P<0.05)巨噬细胞的M2极化,伴随IL-10、CCL-18和VEGF-A分泌量的变化。miR-1278靶向抑制磷酸酶SHP-1的表达(约70%,P<0.05),进而促进信号转导与转录激活因子3(STAT3)通路的活性。WP1066(STAT3抑制剂)处理可以抑制低氧肺癌细胞外泌体诱导的M2极化,WP1066和miR-1278 inhibitor均可抑制低氧肺癌外泌体诱生的巨噬细胞条件培养基对肿瘤细胞迁移和内皮细胞血管形成的诱导作用。结论低氧肺癌来源的外泌体携带的miR-1278通过靶向抑制SHP-1的表达,激活STAT3通路,从而诱导巨噬细胞的M2极化,有助于构建免疫抑制微环境,进而促进肿瘤迁移和血管形成。

Objective To study the role and molecular mechanism of exosome microRNA(miR)-1278 derived from hypoxic lung cancer cells in regulating M2 polarization of macrophages.Methods The culture supernatants of normal and hypoxic lung cancer cells were collected,and the exosomes were separated by differential centrifugation,observed and counted by transmission electron microscope,and the expression level of exosomal miR-1278 was detected by real-time quantitative polymerase chain reaction(PCR).Collected exosomes(10μg)were used to treat CD14+monocytes(mainly macrophages)to induce cell M2 differentiation,and the differentiation extent was evaluated by flow cytometry.MiR-1278 mimic,miR-1278 inhibitor and their negative controls were transfected into CD14+monocytes to evaluate the role of miR-1278 in the differentiation of M2 macrophages.The secretion of interleukin-10(IL-10),chemokine(C-C motif)ligand-18(CCL-18)and vascular endothelial growth factor-A(VEGF-A)were detected by enzyme linked immunosorbent assay(ELISA).The target gene of miR-1278 was predicted,screened and verified,and the regulatory effect of miR-1278 on the target gene and its downstream pathway was investigated.The exosomes from normoxic and hypoxic CL1-5 lung cancer cells were treated with the pathway inhibitor WP1066 or miR-1278 inhibitor,respectively.The proportion of M2 macrophages was detected,and the effects of macrophage conditioned medium on lung cancer cell migration and endothelial cell angiogenesis were studied.Student′s t test was used to analyze the differences between the two groups,and analysis of variance was used to compare the data differences between multiple groups.Results Compared with normoxic lung cancer cells,the number of exosomes secreted by hypoxic lung cancer cells were increased significantly(upregulated 0.8-1.5 folds,P<0.05).Overexpression and inhibition of miR-1278 promoted normoxic(12.06%vs.38.21%,P<0.05)and inhibited(30.55%vs.9.53%,P<0.05)hypoxic M2 polarization of macrophages,respectively,accompanied by changes in the secretion of IL-10,CCL-18 and VEGF-A.MiR-1278 targeted and inhibited the expression of phosphatase SHP-1(about 70%,P<0.05)and promoted activation of the signal transducer and activators of transcription 3(STAT3)pathway.WP1066(STAT3 inhibitor)treatment could inhibit M2 polarization induced by exosomes derived from hypoxic lung cancer cells.Both WP1066 and miR-1278 inhibitor can inhibit tumor cell migration and endothelial cell angiogenesis induced by hypoxic lung cancer exosome-generated conditioned medium in macrophages.Conclusion MiR-1278 carried by hypoxic lung cancer cell exosomes can target and inhibit the expression of SHP-1 and activate the STAT3 pathway,so as to induce M2 polarization of macrophages,build an immunosuppressive microenvironment,and promote tumor migration and angiogenesis.