M2 Macrophage-derived Exosome miR-1246 Regulates the Growth and Invasion of Gastric Cancer Cells
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摘要:
目的 探讨M2巨噬细胞来源的外泌体miR-1246对胃癌AGS细胞增殖，凋亡和侵袭的影响。 方法 采用IL-4和IL-13诱导M2巨噬细胞后，分离其外泌体，并通过透射电镜和免疫印迹法进行鉴定。M2巨噬细胞分别转染NC inhibitor和miR-1246 inhibitor后，分离对应外泌体与AGS细胞共培养，并采用CCK-8，Annexin V-FITC/PI和Transwell分别检测AGS细胞增殖，凋亡和侵袭。TargetScan数据库预测miR-1246下游靶标，并通过双荧光素酶报告基因实验对miR-1246和GSK3B的靶向关系进行验证。 结果 M2巨噬细胞中分离的外泌体大小为50～150 nm，且表达ALIX，CD63和TSG101。M2巨噬细胞来源外泌体增加AGS细胞活力（P < 0.05）和侵袭细胞数（P < 0.01），并降低其凋亡比例（P < 0.01）。敲低外泌体中miR-1246的表达，AGS细胞的表型变化得到回复（P < 0.01）。外泌体miR-1246靶向GSK3B，并调控β-catenin和c-Myc的表达，M2巨噬细胞来源外泌体miR-1246靶向GSK3B促进胃癌细胞增殖和侵袭、并抑制其凋亡（P < 0.001）。 结论 M2巨噬细胞来源外泌体miR-1246靶向GSK3B介导Wnt通路激活促进胃癌细胞增殖和侵袭，并抑制其凋亡。 Abstract:Objective To investigate the effects of M2 macrophage-derived exosome miR-1246 on the proliferation, apoptosis and invasion of AGS cells. Methods After induction of M2 macrophages using LPS and IFN-γ, their exosomes were isolated and identified by transmission electron microscopy and western blot. After M2 macrophages were transfected with NC inhibitor and miR-1246 inhibitor respectively, the corresponding exosomes were isolated and co-cultured with AGS cells, and proliferation, apoptosis, and invasion of AGS cells were detected by CCK-8, Annexin V-FITC/PI and Transwell, respectively. TargetScan database predicted miR-1246 downstream targets and the targeting relationship between miR-1246 and GSK3B was validated by dual-luciferase reporter gene assays. Results The exosomes isolated from M2 macrophages were 30-150 nm in size and expressed ALIX, CD63 and TSG101. M2 macrophage-derived exosomes increased the viability and invasive cell count of AGS cells and decreased their apoptotic ratio. Phenotypic changes in AGS cells were reverted by knocking down the expression of miR-1246 in exosomes. Exosome miR-1246 targets GSK3B and upregulated the expression of β-catenin and c-Myc. Conclusion M2 macrophage-derived exosome miR-1246 mediates Wnt pathway activation to promote proliferation and invasion of gastric cancer cells and inhibit their apoptosis via targeting GSK3B. -
Key words:
- Gastric cancer /
- Macrophages /
- Exosomes /
- miR-1246 /
- Wnt signaling pathway
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图 1 成功分离M2巨噬细胞来源外泌体
A：采用WB检测M2巨噬细胞中标志物（INOS和CD86）的表达水平；B：M2巨噬细胞来源外泌体的透射电镜代表性图片（×1 000）；C：WB检测M2巨噬细胞上清液和分离的囊泡中外泌体标志物（ALIX，CD63和TSG101）的表达水平。相较于Control组，**P < 0.01。
Figure 1. Exosomes derived from M2 macrophages were successfully isolated
图 2 M2巨噬细胞来源外泌体促进AGS细胞增殖和侵袭，并抑制其凋亡
A：CCK-8检测M2巨噬细胞来源外泌体对AGS细胞活力的影响；B：Annexin V-FITC/PI检测AGS细胞凋亡的流式结果；C：Transwell检测AGS细胞侵袭的代表性图片和统计分析（×40）；D：RT-qPCR检测M2巨噬细胞来源外泌体对AGS细胞中miR-1246表达的影响；E：Western blot 检测凋亡相关蛋白claved-caspase3，BAX以及BCL2表达。相较于NC组，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 2. M2 macrophage-derived exosomes promoted the proliferation and invasion of AGS cells，and inhibited their apoptosis
图 3 M2巨噬细胞来源外泌体miR-1246促进AGS细胞增殖和侵袭，并抑制其凋亡
A：RT-qPCR检测各组AGS细胞中miR-1246的表达变化；B：由CCK-8试剂盒检测得到的AGS细胞活力变化；C：Western blot 检测凋亡相关蛋白claved-caspase3，BAX以及BCL2表达；D：AGS细胞凋亡比例的流式代表性图片和统计分析结果；E：Transwell检测AGS细胞的侵袭变化（×40）。相较于NC组，*P < 0.05，**P < 0.01，***P < 0.001；相较于M2-exo/NCinhibitor组，#P < 0.05，##P < 0.01，###P < 0.001。
Figure 3. M2 macrophage-derived exosome miR-1246 promoted the proliferation and invasion of AGS cells，and inhibited their apoptosis
图 4 miR-1246靶向调控Wnt信号通路
A：TargetScan数据库预测得到的miR-1246与GSK3B的潜在结合序列；B：双荧光素酶报告基因实验验证miR-1246与GSK3B的靶向关系；C：WB检测M2巨噬细胞来源外泌体miR-1246对AGS细胞中GSK3B蛋白表达的影响；D：WB检测M2巨噬细胞来源外泌体miR-1246对AGS细胞中Wnt信号通路的影响。相较于NCmimic组，**P < 0.01；相较于NC组，aP < 0.05，aaP < 0.01；相较于M2-exo/NCinhibitor组，bP < 0.05，bbP < 0.01；ns表示差异无统计学意义。
Figure 4. miR-1246 targets the Wnt signaling pathway
图 5 M2巨噬细胞来源外泌体miR-1246靶向GSK3B促进胃癌细胞增殖和侵袭、并抑制其凋亡
A：由CCK-8试剂盒检测得到的AGS细胞活力变化；B：AGS细胞凋亡比例的统计分析结果；C：Transwell检测AGS细胞的侵袭变化统计分析结果；D：AGS细胞凋亡比例的流式代表性图片；E：Transwell检测AGS细胞的侵袭变化代表性图片（×40）；F：Western blot 检测凋亡相关蛋白claved-caspase3，BAX以及BCL2表达。相较于NC组，aP < 0.05，aaP < 0.01，aaaP < 0.001；相较于M2-exo/NCinhibitor组，bP < 0.05，bbP < 0.01；ns表示差异无统计学意义。
Figure 5. M2 macrophage-derived exosome miR-1246 targets GSK3B to promote proliferation and invasion and inhibit apoptosis of gastric cancer cells
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