Zhang Yu-Jie, Cao Qi-Hui, Yang Yong-Hong
Department of Laboratory Medicine, Beijing Jishuitan Hospital Guizhou Hospital, Guiyang 550014, Guizhou Province, China.
Department of Orthopedics, Beijing Jishuitan Hospital Guizhou Hospital, Guiyang 550014, Guizhou Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2024 Oct;32(5):1455-1462. doi: 10.19746/j.cnki.issn.1009-2137.2024.05.024.
To investigate the effect of macrophage polarization mediated by Jumonji domain containing-3 (JMJD3)-interferon regulatory factor 4 (IRF4) signaling pathway on the malignant biological behavior of multiple myeloma (MM) cells.
THP-1 monocytes were induced to differentiate into macrophages by phorbol myristate acetate (PMA). THP-1 macrophages were divided into control group (normal culture), M2 induction group ［added recombinant human interleukin (IL) -4, IL-13 proteins］, M2+JMJD3 protein group (added recombinant human IL-4, IL-13 and JMJD3 proteins) and M2+JMJD3 inhibitor group (added recombinant human IL-4, IL-13 proteins and JMJD3 inhibitor), the proportion of CD206 cells was detected by flow cytometry, the levels of IL-10 and transforming growth factor-β (TGF-β) in the culture supernatant were detected by ELISA assay, the expression levels of arginase-1 and mRNA were detected by real-time quantitative PCR (qRT-PCR), and the expression levels of Arg-1, JMJD3 and IRF4 proteins were detected by Western blot. Correspondingly, human MM cells U266 were cultured with THP-1 macrophage culture supernatant of each group, Methyl thiazolyl tetrazolium (MTT) method and plate colony formation assay were used to detect cell proliferation, cell apoptosis was detected by flow cytometry, Western blot was used to detect the expression levels of apoptosis-promoting protein Bcl-2-associated X protein (Bax) and cleaved caspase-3 in cells, and Transwell assay was used to detect cell migration and invasion.
Compared with the control group, the proportion of CD206 cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2 induction group were significantly increased ( <0.001), meanwhile, the proliferation activity and the number of clones of U266 cells were significantly increased ( <0.01), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased ( <0.001), the numbers of migrated cells and invasive cells were increased ( <0.001). Compared with M2 induction group, the proportion of CD206 cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2+JMJD3 protein group were further increased ( <0.01), meanwhile, the proliferation activity and the number of clones of U266 cells were further increased ( <0.05), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were further decreased ( <0.01), the numbers of migrated cells and invasive cells were further increased ( <0.001); However, the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group.
M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation, migration and invasion of MM cells, and inhibit cell apoptosis.
探讨含Jumonji结构域蛋白3（JMJD3）-干扰素调节因子4（IRF4）信号通路介导的巨噬细胞极化对多发性骨髓瘤（MM）细胞恶性生物学行为的影响。
用佛波酯（PMA）诱导THP-1单核细胞分化为巨噬细胞。将THP-1巨噬细胞分为对照组（正常培养）、M2诱导组［加入重组人白细胞介素（IL）-4、IL-13蛋白］、M2+JMJD3蛋白组（加入重组人IL-4、IL-13及JMJD3蛋白）和M2+JMJD3抑制剂组（加入重组人IL-4、IL-13蛋白及JMJD3抑制剂），采用流式细胞术检测CD206细胞比例，ELISA法检测培养上清中IL-10和转化生长因子-β（TGF-β）水平，实时定量PCR（qRT-PCR）检测精氨酸酶-1 及mRNA表达水平，Western blot检测Arg-1、JMJD3和IRF4蛋白表达水平。相应地，将人MM细胞U266与各组THP-1巨噬细胞培养上清共同培养，采用甲基噻唑基四氮唑（MTT）法和平板克隆形成试验检测细胞增殖，流式细胞术检测细胞凋亡，Western blot检测细胞中促凋亡蛋白Bcl-2相关X蛋白（Bax）和裂解的半胱天冬酶-3表达水平，Transwell试验检测细胞迁移和侵袭能力。
与对照组比较，M2诱导组THP-1巨噬细胞中CD206细胞比例、Arg-1、JMJD3和IRF4的mRNA及蛋白表达水平、细胞培养上清中IL-10和TGF-β水平均显著升高（<0.001），同时U266细胞增殖活性及克隆数显著增加（<0.01），凋亡率及促凋亡蛋白Bax和裂解的半胱天冬酶-3表达水平显著降低（<0.001），迁移细胞数和侵袭细胞数增加（<0.001）。与M2诱导组比较，M2+JMJD3蛋白组THP-1巨噬细胞中CD206细胞比例、Arg-1、JMJD3和IRF4的mRNA及蛋白表达水平、细胞培养上清中IL-10和TGF-β水平进一步升高（<0.01），同时U266细胞增殖活性及克隆数进一步增加（<当0.05），凋亡率及促凋亡蛋白Bax和裂解的半胱天冬酶-3表达水平进一步降低（<0.01），迁移细胞数和侵袭细胞数进一步增加（<0.001）；而M2+JMJD3抑制剂组上述指标变化趋势与M2+JMJD3蛋白组相反。
JMJD3-IRF4信号通路介导的巨噬细胞M2极化可促进MM细胞增殖、迁移和侵袭，抑制细胞凋亡。
