| 李 登,沙明磊,陈 磊,赵 圣,邵 怡.miR-203下调TLR4-Myd88-NF-κB信号通路诱导M2型巨噬细胞极化[J].现代生物医学进展英文版,2018,(17):3201-3208. |
| miR-203下调TLR4-Myd88-NF-κB信号通路诱导M2型巨噬细胞极化 |
| miR-203 Regulating M2 Macrophage Polarization Via Inhibition of TLR4-Myd88-NF-κB Signaling Pathway |
| Received:March 15, 2018 Revised:March 31, 2018 |
| DOI：10.13241/j.cnki.pmb.2018.17.001 |
| 中文关键词: miR-203 RAW264.7 双荧光素酶报告基因系统 巨噬细胞极化 TLR4-Myd88-NF-κB |
| 英文关键词: miR-203 RAW264.7 Dual-luciferase reporter assay system Macrophage polarization TLR4-Myd88-NF-κB |
| 基金项目:国家自然科学基金项目(81200504)；上海交通大学医工交叉项目[多学科交叉项目培育（医工）]（YG2016MS20） |
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| 中文摘要: |
| 摘要 目的：验证miR-203通过与TLR4 mRNA的3'-UTR特异性结合下调TLR4-Myd88-NF-κB信号通路诱导M2型巨噬细胞极化。方法：通过miRanda软件预测TLR4 mRNA 3'-UTR存在miR-203结合位点。根据TLR4 mRNA 3'-UTR序列设计目的基因片段以及突变型目的基因片段，以pmirGLO为载体构建双荧光素酶报告基因野生型载体(pmirGLO-TLR4 3'-UTR)及其突变型载体(pmirGLO-mut-TLR4 3'-UTR)。将293T细胞共转染pmirGLO-TLR4 3'-UTR质粒或pmirGLO-mut-TLR4 3'-UTR质粒及mmu-miR-203-3p mimics或mimic NC，通过双荧光素酶报告基因系统验证miR-203可以与TLR4 mRNA的3'-UTR特异性结合，通过Real-time PCR及Western blot验证小鼠巨噬细胞RAW264.7转染了mmu-miR-203-3p mimics、mimic NC后，M1型巨噬细胞markers (iNOS, TNF-α, CCL-3, IL-23)，M2型巨噬细胞markers (Arg-1, CX3CR1, IL-4, MRC, IL-10, Ym-1)及TLR4-Myd88-NF-κB信号通路的表达。使用TLR4抑制剂TAK-242抑制小鼠巨噬细胞TLR4-Myd88-NF-κB信号通路后，转染mmu-miR-203-3p mim- ics、mimic NC，再次检测M1，M2型巨噬细胞markers及TLR4-Myd88-NF-κB信号通路的表达。结果：双荧光素酶报告基因系统显示293T细胞转染了pmirGLO-TLR4 3'-UTR质粒及mmu-miR-203-3p mimics后，其相对荧光素酶活性△CT较转染了pmirG- LO-mut-TLR4 3'-UTR质粒或者mimic NC均有显著降低，其差异达到统计学意义（P<0.05）。Real-time PCR及Western blot显示转染了mmu-miR-203-3p mimics的小鼠巨噬细胞M1型巨噬细胞markers (iNOS, TNF-α, CCL-3, IL-23)表达减少，M2型巨噬细胞markers (Arg-1, CX3CR1, IL-4, MRC, IL-10, Ym-1)表达增加，同时伴有TLR4-Myd88-NF-κB信号通路表达下调，而通过给予TAK-242抑制了mmu-miR-203-3p mimics下调TLR4-Myd88-NF-κB信号通路及诱导M2型巨噬细胞极化的作用。结论：miR-203与TLR4 mRNA的3'-UTR特异性结合下调TLR4-Myd88-NF-κB信号通路诱导M2型巨噬细胞极化。 |
| 英文摘要: |
| ABSTRACT Objective: To verify that miR-203 plays a significant role in promoting M2 macrophage polarization through targeting 3'-UTR of TLR4 mRNA and therefore inhibiting TLR4-Myd88-NF-κB signaling. Methods: We speculated the specific binding of miR-203 and 3'-UTR of TLR4 mRNA by miRanda software. And we constructed plasmid pmirGLO-TLR4 3'-UTR and its mutant plas- mid pmirGLO-mut-TLR4 3'-UTR based on the sequence of 3'-UTR of TLR4 mRNA. Cell line 293T was co-transfected with plasmids pmirGLO-TLR4 3'-UTR or pmirGLO-mut-TLR4 3'-UTR and mmu-miR-203-3p mimics or mimic NC. Dual-luciferase reporter assay sys- tem was used to detect the specific binding of miR-203 and 3'-UTR of TLR4 mRNA. The expressions of M1 macrophage markers (iNOS, TNF-α, CCL-3, IL-23), M2 macrophage markers (Arg-1, CX3CR1, IL-4, MRC, IL-10 and Ym1) were detected by Real-time PCR. And the expression of TLR4-Myd88-NF-κB signaling was detected by Real-time PCR and Western blot. The antagonist of TLR4, TAK-242, was used to further confirmed the role of miR-203 in regulating M2 macrophage polarization and inhibiting the TLR4-Myd88-NF-κB sig- naling pathway. Results: The Relative luciferase activity was significantly lower in 293T cells co-transfected with pmirGLO-TLR4 3'-UTR plasmid and mmu-miR-203-3p mimics than in 293T cells co-transfected with either pmirGLO-mut-TLR4 3'-UTR plasmid or mimic NC. Real-time PCR and Western blot revealed that the expressions of M1 macrophage markers (iNOS, TNF-α, CCL-3, IL-23) were significantly decreased and the expressions of M2 macrophage markers (Arg-1, CX3CR1, IL-4, MRC, IL-10 and Ym1) were significantly increased in mouse macrophage RAW264.7 transfected with mmu-miR-203-3p mimics when compared with control group or mouse macrophage RAW264.7 transfected with mimic NC, accompanied by downregulation of TLR4-Myd88-NF-κB signaling. And this phenomenon was abrogated by the inhibiting of TLR4-Myd88-NF-κB signaling pathway in mouse macrophages RAW264.7 treated with TAK-242. Conclusion: Taken together, miR-203 could promote M2 macrophages polarization through directly targeting 3'-UTR of TLR4 mRNA and therefore inhibiting the TLR4-Myd88-NF-κB signaling pathway. |
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