| Objective: Interleukin-1?(IL-1?)is an inflammatory cytokine and is closely relatedto the occurrence and development of tumors.Innate immune cells are the main sourceof IL-1? secretion,and macrophages are the main ones.Most of macrophages in thetumor microenvironment are educated into M2-type tumor associated macrophages(TAMs).Some studies have shown that TAMs are an accomplice to tumor progression,but whether TAMs secrete IL-1? is still unknown.In the previous study,we had foundthat tumor cells derived microparticles(T-MPs)induced the differentiation ofmacrophages toward M2 phenotype.So,did the T-MPs play a role in the secretion ofIL-1? by TAMs? This study was to investigate whether the microparticles derived fromlung cancer cells induce macrophage up-regulation of IL-1?.On this basis,themolecular mechanism of IL-1? production and the reasons for tumor developmentinduced by IL-1? will be explored.Methods: 1.Macrophages were treated with L-MPs for 12 h,cells were collected and RNA was extracted for real-time PCR analysis including arginase-1,VEGF,IL-10 and IL-1? gene.2.Macrophages were treated with RNA or DNA extracted from LMPs for 12 h,IL-1? m RNA level was analyzed by real-time PCR.3.Macrophages were transfected with TLR3 si RNA and then treated with L-MPs,12 h later,IL-1? m RNA level was analyzed by real-time PCR.4.Macrophages were treated with L- MPs.And then cells were collected and the expression of phosphorylation of MAPK and NF-?B signal protein was detected by western blot.5.A comprehensive sequencing of L-MPs RNA and tumor cell RNA were analyzed by Hi Seq2500 system.The sequences of two RNA samples were compared.6.Macrophages were treated with L-MPs,and then the expression of active caspase-1 was detected by western blot.7.Macrophages were treated with L-MPs,and then were loaded with Cell ROX or Mitosox to analyze cell or mitochondrial ROS level.8.Macrophages were treated with L-MPs,and then were loaded with Fluo-4 AM to analyze cell Ca2+ level.9.Lewis cell muscle transporting models were injected of L-MPs in tumor,and then IL-1? secreted by macrophages which took up L-MPs were analyzed.The effect of IL-1? to tumor growth were evaluated.10.Humanized mice tumor model were injected of L-MPs in tumor.IL-1? level in macrophages phagocytizing L-MPs were analyzed,and the effect of IL-1? to tumor growth was evaluated.11.The isolated tumor cells from cancer model were seeded into the soft 3D fibrin gels to screen TRCs.The TRCs colony size was analyzed.Results: 1.L-MPs upregulated arginase-1,VEGF,IL-10 and IL-1? mRNA expression in macrophages.2.TLR3 activation mediated phosphorylation of MAPK and NF-?B signal protein by L-MPs,and then IL-1? m RNA expression was upregulated.3.Non-coding RNAs enriched in L-MPs are potential ligands for TLR3.4.L-MPs induce mitochondrial ROS to activate NLRP3 and caspase-1 for IL-1? cleavage.5.Lysosomal calcium release by L-MPs caused mitochondrial ROS production.6.L-MPs-induced IL-1? by macrophages promoted TRCs growth and lung tumor development.7.L-MP-induced IL-1? by macrophages facilitated tumor growth in a humanized mouse model.Conclusions: L-MPs promoted pro-IL-1? expression through TLR3 signal via RNA segments.L-MPs mediated lysosomal Ca2+ release to cytoplasm and then induced mitochondrial ROS generation which could activate inflammasome and caspase-1.Finally,activated caspase-1 cleaves pro-IL-1? to produce active IL-1?.IL-1? upregulation mediated stemness induction of tumor cells and then facilitated tumor development. |
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