JMJD3-IRF4信号通路介导的巨噬细胞极化对多发性骨髓瘤细胞恶性生物学行为的影响

Effect of JMJD3-IRF4 Signaling Pathway Mediated Macrophage Polarization on the Malignant Biological Behavior of Multiple Myeloma Cells

目的: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。方法: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(添加重组人IL-4、IL-13蛋白)、M2+JMJD3蛋白组(添加重组人IL-4、IL-13和JMJD3蛋白)和M2+JMJD3抑制剂组(添加重组人IL-4、IL-13蛋白和JMJD3抑制剂),流式细胞术检测CD206^(+)细胞比例,ELISA检测培养上清液中IL-10、转化生长因子-β(TGF-β)水平,实时荧光定量PCR(qRTPCR)检测细胞中精氨酸酶-1(Arg-1)、JMJD3、IRF4 mRNA表达水平,免疫印迹(Western blot)检测Arg-1、JMJD3、IRF4蛋白表达水平。相应地,使用各组THP-1巨噬细胞培养上清液培养人MM细胞U266,MTT法、平板克隆形成实验检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,Western blot检测细胞中促凋亡蛋白Bcl-2相关X蛋白(Bax)、活化的半胱氨酸天冬氨酸蛋白水解酶-3(cleaved caspase-3)表达水平,Transwell实验检测细胞迁移、侵袭情况。结果: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、IRF4 mRNA和蛋白表达水平以及细胞培养上清液中IL-10、TGF-β水平明显升高(P<0.001),同时U266细胞增殖活力、集落形成数明显增加(P<0.01),细胞凋亡率以及促凋亡蛋白Bax、cleaved caspase-3表达水平明显降低(P<0.001),迁移、侵袭细胞数明显增多(P<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、IRF4 mRNA和蛋白表达水平以及细胞培养上清液中IL-10、TGF-β水平均进一步升高(P<0.01),同时U266细胞增殖活力、集落形成数明显升高(P<0.05),细胞凋亡率以及促凋亡蛋白Bax、cleaved caspase-3表达水平明显降低(P<0.01),迁移、侵袭细胞数明显增多(P<0.001);而M2+JMJD3抑制剂组上述各指标变化趋势与M2+JMJD3蛋白组相反。结论:JMJD3-IRF4信号通路介导的巨噬细胞M2极化能够促进MM细胞增殖、迁移和侵袭,并抑制细胞凋亡。

Objective: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.Methods: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(L)4,L-13 proteins],M2+JMJD3 protein group(added recombinant human IL4,IL-13 and JMJD3 proteins)and M2+JMJD3 inhibitor group(added recombinant human IL4,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-B(TGF-β)in the culture supernatant were detected by ELISA assay,the expression levels of arginase-1.(Arg-l),JMJD3 and IRF4 mRNA were detected by real-time quantitative PCR(qRT-PCR),and the expression levels of Arg-1,JMJD3 and IRF4 proteins were detected by W estern 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.Results: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-βin the cell culture supernatant in M2 induction group were significantly increased(P<0.001),meanwhile,the proliferation activity and the number of clones of U266 cells were significantly increased(P<0.01),the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased(P<0.001),the numbers of migrated cells and invasive cells were increased(P<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-βin the cell culture supernatant in M2+JMJD3 protein group were further increased(P<0.01),meanwhile,the proliferation activity and the number of clones of U266 cells were further increased(P<0.05),the apoptosis rate and the expression levels of apoptosis promoting protein Bax and cleaved caspase-3 were further decreased(P<0.01),the numbers of migrated cells and invasive cells were further increased(P<0.001);However,the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group.Conclusion:M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation,migration and invasion of MM cells,and inhibit cell apoptosis.