Yang X, Shen H, Li Q, Dai Z, Yang R, Huang G, Chen R, Wang F, Song J, Hua H
Department of Pathology and Pathophysiology, Kunming Medical University, Kunming 650500, China.
Department of Pathology, Zhaotong First People's Hospital, Zhaotong 657099, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 May 20;42(5):658-664. doi: 10.12122/j.issn.1673-4254.2022.05.05.
To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells.
C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment.
The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (=0.001), than the brain tissues of the control mice ( < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs ( < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA( < 0.01)and protein ( < 0.01, < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells ( < 0.05).
Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.
探讨干扰肿瘤相关巨噬细胞（TAM）中P2X4受体表达对胶质瘤细胞侵袭和迁移的影响。
对尾状核接种胶质瘤的C57BL/6小鼠模型进行HE染色观察胶质瘤病理情况，采用免疫荧光法检测Iba-1和P2X4受体的表达。用GL261细胞的条件培养基将RAW264.7细胞诱导为TAM，采用RT-qPCR和蛋白质印迹法检测巨噬细胞极化相关标志物的mRNA表达变化以及P2X4受体的mRNA和蛋白表达。用RT-qPCR和蛋白质印迹法检测siRNA介导的P2X4干扰对TAM中IL-1β和IL-18 mRNA及蛋白表达的影响。将GL261细胞培养于转染后的TAM的条件培养基中，采用Transwell侵袭和迁移实验评估细胞的侵袭和迁移能力。
荷瘤小鼠的胶质瘤组织中Iba-1阳性细胞数量明显多于对照小鼠的脑组织（<0.001），且检测到P2X4受体表达明显增加（=0.001）。RAW264.7细胞来源的TAM中M2巨噬细胞标志物（Arg-1和IL-10）和M1巨噬细胞标志物（iNOS和TNF-α）均显著上调（均<0.01），但M2巨噬细胞标志物的上调更明显；TAM中P2X4受体蛋白和mRNA表达水平均升高（<0.05）。干扰P2X4受体表达可显著降低TAM中IL-1β和IL-18的mRNA（<0.01）和蛋白（<0.01，<0.05）表达水平，并明显抑制TAM促进胶质瘤细胞侵袭和迁移的能力（<0.05）。
干扰TAM中的P2X4受体可能通过降低IL-1β和IL-18的表达来抑制胶质瘤细胞的迁移和侵袭。
