miR-7基因敲减对LPS诱导巨噬细胞炎症反应的影响
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摘要
观察miR-7基因敲减(knockdown, KD)对LPS诱导的巨噬细胞炎症反应的影响,并初步探讨其机制。分别用0、50、100、200 ng/mL LPS刺激野生型(wild type, WT)小鼠骨髓来源巨噬细胞12 h,经实时荧光定量PCR(real-time fluorescence quantitative PCR, Real-time PCR)检测发现,随着LPS刺激浓度的增加,巨噬细胞中miR-7的表达水平逐渐升高,且在100 ng/mL LPS浓度刺激下miR-7表达最显著(P<0.01)。用100 ng/mL LPS处理miR-7 KD小鼠和WT小鼠骨髓来源巨噬细胞12 h,镜检观察到巨噬细胞形态均由圆形变为长梭形,并伸出长长的伪足。应用流式细胞术检测巨噬细胞表面CD80和CD86分子的表达情况,结果显示,LPS作用后,miR-7 KD小鼠骨髓来源巨噬细胞表面CD80和CD86的比例较WT小鼠显著上调(P<0.05)。Real-time PCR和ELISA结果显示,miR-7 KD小鼠巨噬细胞在LPS刺激后,炎性因子IL-1β、IL-6和TNF-ɑ的表达显著增加(P<0.05)。Western blotting检测结果显示,LPS刺激后,与对照组相比,miR-7 KD小鼠巨噬细胞中NF-κB p65和p-NF-κB p65的蛋白水平显著升高(P<0.01)。以上数据显示,miR-7基因KD可显著增强LPS诱导的巨噬细胞炎症反应,可能与NF-κB信号通路的传递增强有关,提示miR-7在巨噬细胞炎症过程中发挥重要的调控作用。
To study the effect of miR-7 knockdown(KD) on LPS-induced macrophage inflammation in murine and its molecular mechanism, bone marrow-derived macrophages from wild type(WT) mice were stimulated with 0, 50, 100, 200 ng/mL LPS for 12 h.The relative expression of miR-7 in macrophages was evaluated by real-time fluorescence quantitative PCR(Real-time PCR).The result showed that the expression of miR-7 in macrophages gradually increased with the increase of LPS concentration and the 100 ng/mL was the most effective(P < 0. 01). Bone marrow-derived macrophages from both WT miceand miR-7 KD mice became long fusiform and stretched out long pseudopodia after LPS stimulation. The proportion of related surface molecules on macrophage, such as CD80 and CD86, was analyzed by flow cytometry. Compared with WT mice, the proportions of CD80 and CD86 on macrophages from miR-7 KD mice were significantly upregulated after LPS stimulation(P < 0.05). The relative expression of inflammatory cytokines, such as IL-1β, IL-6 and TNF-ɑ, in macrophage was detected by Real-time PCR and ELISA. The analysis showed that the levels of IL-1β, IL-6 and TNF-ɑ in miR-7 KD mice increased significantly after LPS stimulation(P < 0.05). The relative expressions of NF-κB p65 and p-NF-κB p65 proteins were detected by Western blotting. Compared with WT mice, the protein levels of NF-κB p65 and p-NF-κB p65 in miR-7 KD mice increased significantly(P < 0.01). Together, miR-7 deficiency could significantly enhance LPS-induced macrophage inflammatory responses, which may be associated with the enhancement of NF-κB signaling pathway. Therefore, miR-7 may play an important regulatory role in macrophage inflammatory process.
To study the effect of miR-7 knockdown(KD) on LPS-induced macrophage inflammation in murine and its molecular mechanism, bone marrow-derived macrophages from wild type(WT) mice were stimulated with 0, 50, 100, 200 ng/mL LPS for 12 h.The relative expression of miR-7 in macrophages was evaluated by real-time fluorescence quantitative PCR(Real-time PCR).The result showed that the expression of miR-7 in macrophages gradually increased with the increase of LPS concentration and the 100 ng/mL was the most effective(P < 0. 01). Bone marrow-derived macrophages from both WT miceand miR-7 KD mice became long fusiform and stretched out long pseudopodia after LPS stimulation. The proportion of related surface molecules on macrophage, such as CD80 and CD86, was analyzed by flow cytometry. Compared with WT mice, the proportions of CD80 and CD86 on macrophages from miR-7 KD mice were significantly upregulated after LPS stimulation(P < 0.05). The relative expression of inflammatory cytokines, such as IL-1β, IL-6 and TNF-ɑ, in macrophage was detected by Real-time PCR and ELISA. The analysis showed that the levels of IL-1β, IL-6 and TNF-ɑ in miR-7 KD mice increased significantly after LPS stimulation(P < 0.05). The relative expressions of NF-κB p65 and p-NF-κB p65 proteins were detected by Western blotting. Compared with WT mice, the protein levels of NF-κB p65 and p-NF-κB p65 in miR-7 KD mice increased significantly(P < 0.01). Together, miR-7 deficiency could significantly enhance LPS-induced macrophage inflammatory responses, which may be associated with the enhancement of NF-κB signaling pathway. Therefore, miR-7 may play an important regulatory role in macrophage inflammatory process.
引文
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[3] Vijay K.Toll-like receptors in immunity and inflammatory diseases:Past,present,and future[J].Int Immunopharmacol,2018,59:391-412.
[4] Martinez-Quiles N,Goldbach-Mansky R.Updates on autoinflammatory diseases[J].Curr Opin Immunol,2018,55:97-105.
[5] Nejad C,Stunden HJ,Gantier MP.A guide to miRNAs in inflammation and innate immune responses[J].FEBS J,2018,285(20):3695-3716.
[6] Saliminejad K,Khorshid HR,Soleymani Fard S,et al.An overview of microRNAs:Biology,functions,therapeutics,and analysis methods[J].J Cell Physiol,2019,234(5):5451-5465.
[7] Kehl T,Backes C,Kern F,et al.About miRNAs,miRNA seeds,target genes and target pathways[J].Oncotarget,2017,8(63):107167-107175.
[8] O'Connell RM,Rao DS,Baltimore D.MicroRNA regulation of inflammatory responses[J].Annu Rev Immunol,2012,30:295-312.
[9] Moein S,Vaghari-Tabari M,Qujeq D,et al.MiRNAs and inflammatory bowel disease:An interesting new story[J].J Cell Physiol,2019,234(4):3277-3293.
[10] Xie WD,Li MN,Xu NH,et al.miR-181a regulates infla-mmation responses in monocytes and macrophages[J].PLoS One,2013,8(3):e58639.
[11] Zhang L,Huang CQ,Guo YJ,et al.MicroRNA-26b modulates the NF-κB pathway in alveolar macrophages by regulating PTEN[J].J Immunol,2015,195(11):5404-5414.
[12] Ballegaard V,Ralfkiaer U,Pedersen KK,et al.MicroRNA-210,MicroRNA-331,and MicroRNA-7 are differentially regulated in treated HIV-1-infected individuals and are associated with markers of systemic inflammation[J].J Acquir Immune Defic Syndr,2017,74(4):e104-e113.
[13] Dong YF,Chen ZZ,Zhao Z,et al.Potential role of micro-RNA-7 in the anti-neuroinflammation effects of nicorandil in astrocytes induced by oxygen-glucose deprivation[J].J Neuroinflammation,2016,13:60.
[14] 朱顺飞,李永菊,陈超,等.MicroRNA-7基因敲减小鼠模型的鉴定[J].遵义医学院学报,2014,37(6):582-586;590.
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[16] Zhao J,Tao Y,Zhou Y,et al.MicroRNA-7:Apromising new target in cancer therapy[J].Cancer Cell Int,2015,15:103.
[17] Kabir TD,Ganda C,Brown RM,et al.A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma[J].Hepatology,2018,67(1):216-231.
[18] Cui YX,Bradbury R,Flamini V,et al.MicroRNA-7 suppresses the homing and migration potential of human endothelial cells to highly metastatic human breast cancer cells[J].Br J Cancer,2017,117(1):89-101.
[19] Zhou Y,Lu M,Du RH,et al.MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson's disease[J].Mol Neurodegener,2016,11:28.
[20] Zhao J,Chen C,Guo M,et al.MicroRNA-7 deficiency ameliorates the pathologies of acute lung injury through elevating KLF4[J].Front Immunol,2016,7:389.
[21] 岳东旭,赵娟娟,胡琳,等.微小RNA-7基因敲减对LPS诱导的脑部炎症的影响[J].现代免疫学,2018,38(5):366-371.
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[23] Wang Z,Wang P,Wang Z,et al.MiRNA-548c-5p downregulates inflammatory response in preeclampsia via targeting PTPRO[J].J Cell Physiol,2019,234(7) :11149-11155.
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[25] Yuan K,Zhang X,Lv L,et al.Fine-tuning the expression of microRNA-155 controls acetaminophen-induced liver infla-mmation[J].Int Immunopharmacol,2016,40:339-346.
[26] Mann M,Mehta A,Zhao JL,et al.An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses[J].Nat Commun,2017,8(1):851.
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