脂多糖诱导巨噬细胞中核受体协同抑制因子启动子甲基化对炎症因子的调控作用

doi:10.3877/cma.j.issn.1674-6902.2017.02.009

目的

探讨核受体协同抑制因子(NCOR)在脂多糖(LPS)诱导巨噬细胞炎症反应中的作用及其调控机制。

方法

1 μg/ml的LPS分别处理小鼠巨噬细胞RAW264.7 24 h和48 h，应用Western blot和Real time-PCR检测NCOR的表达水平以及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6) mRNA水平，荧光素酶报告基因检测核因子-κB (NF-κB)的启动子活性。LPS处理细胞48 h后，应用MSP检测NCOR启动子是否发生甲基化以及Western blot检测DNMT3b的表达变化。Real time-PCR检测5′-aza和LPS联合处理细胞后NCOR mRNA的表达水平；转染DNMT3b siRNA后，分别应用Western blot和Real time-PCR检测DNMT3b的表达水平，以及DNMT3b siRNA和LPS联合作用下NCOR、TNF-α、IL-6的表达水平和NF-κB的启动子活性。

结果

LPS干预细胞24、48 h后，NCOR蛋白和mRNA表达显著下调(P<0.05)，而TNF-α、IL-6 mRNA表达水平、DNMT3b蛋白的表达水平以及NF-κB的启动子活性显著上升(P<0.05)。MSP检测说明LPS可介导NCOR的启动子甲基化。用5′-aza和LPS联合处理细胞后NCOR mRNA水平较LPS组有显著上升(P<0.05)。采用DNMT3b siRNA可显著下调DNMT3b蛋白和mRNA水平，并可部分逆转LPS介导的抑制NCOR表达的效应，抑制TNF-α、IL-6的表达水平和NF-κB的启动子活性(P<0.05)。

结论

NCOR启动子的甲基化是LPS介导巨噬细胞炎症反应发生、发展的关键步骤，其可作为治疗ALI/ARDS的潜在靶点。

关键词: 核受体协同抑制因子, DNA甲基化, 核转录因子κB, 炎症因子

Objective

To investigate the role and potential mechanism of nuclear receptor corepressor (NOCR) at lipopolysaccharide(LPS) mediation inflammation response in macrophages.

Methods

Western blot, realtime-PCR and luciferase assays was used to detect the expression of NCOR, interleukin-6(IL-6) and tumour necrosisfactor(TNF-α) and promoter activity of nuclear factor-κB (NF-κB) when RAW264.7 cells were treated with 1 μg/ml LPS. The promoter methylation of NCOR was analyzed by methylating-specific PCR(MSP) analysis, and the protein of DNMT3b was evaluated by western blot after cells stimulation with 1 μg/ml LPS for 48 h. Real time-PCR was also used to evaluate the expression of NCOR mRNA when RAW264.7 was treated with 1 μg/ml LPS combine with 1μM 5′-aza. After DNMT3b siRNA was transfected into RAW264.7 for 48 h, the mRNA and protein of DNMT3b was detected by western blot and real time-PCR. Additional, the expression of NCOR, TNF-α and IL-6 and NF-κB activity was analyzed after RAW264.7 was treated with 1 μg/ml LPS combine with DNMT3b siRNA.

Results

The expression of NCOR mRNA and protein was significantly decreased (P<0.05) and the expression of TNF-α and IL-6 mRNA, DNMT3b protein and activity of NF-κB was elevated (P<0.05) after cells was treated with 1 μg/ml LPS for 24 and 48 h, respectively. MSP assay showed LPS could modulate the NCOR promoter methylation. The expression of DNMT3b mRNA and protein was decreased after cells were transfected by DNMT3b siRNA. Additionally, down-regulation of DNMT3b was partly reversed LPS modulation the inhibition of NCOR, and decreased the expression of TNF-α and IL-6 mRNA, and activity of NF-κB (P<0.05).

Conclusion

NCOR promoter methylation is key step of occurrence and development of LPS mediation inflammation response. It might be a potential target for acute lung injury / acute respiratory distress syndrome (ALI/ARDS) treatment.

Key words: Nuclear receptor corepressor, DNA methylation, Nuclear factor-k-gene binding protein, Inflammatory factor

表1 基因引物序列

基因名称	序列(5′→3′)
NCOR	上游：5′-ATTGGAACGATACAGAGAAGATT-3′
?	下游：5′-GGAACGCTTCACGAATTTG-3′
DNMT3b	上游：5′-ACACTCCAGCTGGGTGTATGCCCTAAC-3′
?	下游：5′-CTCAACTGGTGTCGTGGAGT-3′
IL-6	上游：5′-GTATGAACAACGATGATGCACTTG-3′
?	下游：5′-ATGGTACTCCAGAAGACCAGAGGA-3′
TNF-α	上游：5′-GACCCTCACACTCAG ATCATC-3′
?	下游：5′- GAACCTGGGAGTAGATAAGG-3′

表1 基因引物序列

基因名称	序列(5′→3′)
NCOR	上游：5′-ATTGGAACGATACAGAGAAGATT-3′
?	下游：5′-GGAACGCTTCACGAATTTG-3′
DNMT3b	上游：5′-ACACTCCAGCTGGGTGTATGCCCTAAC-3′
?	下游：5′-CTCAACTGGTGTCGTGGAGT-3′
IL-6	上游：5′-GTATGAACAACGATGATGCACTTG-3′
?	下游：5′-ATGGTACTCCAGAAGACCAGAGGA-3′
TNF-α	上游：5′-GACCCTCACACTCAG ATCATC-3′
?	下游：5′- GAACCTGGGAGTAGATAAGG-3′

图1 LPS处理RAW264.7细胞后NCOR、IL-6和TNF-α表达以及NF-κB活性的变化，1 μg/ml LPS分别处理RAW264.7细胞0 h、24 h、48 h；注：A. Western blot检测NCOR蛋白的表达水平；B: Real time-PCR检测NCOR mRNA的表达水平；^*与0 h比较，P<0.05；C：荧光素酶报告基因检测NF-κB启动子活性；^*与0 h比较，P<0.05，^{^}与24 h比较，P<0.05；D: Real time-PCR检测IL-6和TNF-α mRNA的表达水平；^*与0 h比较，P<0.05，^{^}与24 h比较，P<0.05

图2 LPS处理RAW264.7细胞对NCOR启动子甲基化的影响；注：A：MSP检测1 μg/ml LPS处理细胞48 h后，NCOR启动子甲基化情况。U代表非甲基化NCOR DNA片段；M代表甲基化NCOR DNA片段；B：Real time-PCR检测NCOR mRNA的表达水平。^*与control组比较，P<0.05；^与LPS组比较，P<0.05；C: Western blot检测1 μg/ml LPS处理细胞48 h后DNMT3b蛋白的表达水平

图3 下调DNMT3b对NCOR表达的影响；注：A和B: Real time-PCR和western blot检测DNMT3b siRNA及NC转染RAW264.7细胞后，NCOR mRNA和蛋白的表达水平，^*与NC组比较，P<0.05；C和D: DNMT3b siRNA转染24 h后再用1 μg/ml LPS处理24 h，Real time-PCR和western blot检测NCOR mRNA和蛋白的表达水平，^*与NC组比较，P<0.05；^{^} DNMT3b siRNA组比较，P<0.05；^# NC＋LPS组比较，P<0.05

图4 下调DNMT3b对NF-κB活性及IL-6和TNF-α表达的影响；注：A:荧光素酶报告基因检测NF-κB启动子活性；B: Real time-PCR检测IL-6和TNF-α mRNA的表达水平，^*与NC组比较，P<0.05；^{^} DNMT3b siRNA组比较，P<0.05；^#NC＋LPS组比较，P<0.05

1	宋旸，蒋昊翔，张永红，等. 急性呼吸窘迫综合征药物研究进展[J/CD]. 中华肺部疾病杂志(电子版), 2015, 8(6): 769-772.
2	Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury[J]. N Engl J Med, 2005, 353(16): 1685-1693.
3	Zambon M, Vincent JL. Mortality rates for patients with acute lung injury/ARDS have decreased over time[J]. Chest, 2008, 133(5): 1120-1127.
4	Villar J, Sulemanji D, Kacmarek RM. The acute respiratory distress syndrome: incidence and mortality, has it changed？[J]. Curr Opin Crit Care, 2014, 20(1): 3-9.
5	Opal SM, Dellinger RP, Vincent JL, et al. The next generation of sepsis clinical trial designs: what is next after the demise of recombinant human activated protein C？*[J]. Crit Care Med, 2014, 42(7): 1714-1721.
6	Matthay MA, Ware LB, Zimmerman GA. The acute respiratory distress syndrome[J]. J Clin Invest, 2012, 122(8): 2731-2740.
7	Hong-Wei G, Lan L, De-Guo X, et al. NCoR negatively regulates adipogenic differentiation of mesenchymal stem cells[J]. In Vitro Cell Dev Biol Anim, 2015, 51(7): 749-758.
8	Wong MM, Guo C, Zhang J. Nuclear receptor corepressor complexes in cancer: mechanism, function and regulation[J]. Am J Clin Exp Urol, 2014, 2(3): 169-187.
9	Heldring N, Nyman U, Lönnerberg P, et al. NCoR controls glioblastoma tumor cell characteristics[J]. Neuro Oncol, 2014, 16(2): 241-249.
10	Jennewein C, Kuhn AM, Schmidt MV, et al. Sumoylation of peroxisome proliferator-activated receptor gamma by apoptotic cells prevents lipopolysaccharide-induced NCoR removal from kappaB binding sites mediating transrepression of proinflammatory cytokines[J]. J Immunol, 2008, 181(8): 5646-5652.
11	Barrès R, Yan J, Egan B, et al. Acute exercise remodels promoter methylation in human skeletal muscle[J]. Cell Metab, 2012, 15(3): 405-411.
12	Crevillén P, Yang H, Cui X, et al. Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state[J]. Nature, 2014, 515(7528): 587-590.
13	Qiu W, Baccarelli A, Carey VJ, et al. Variable DNA methylation is associated with chronic obstructive pulmonary disease and lung function[J]. Am J Respir Crit Care Med, 2012, 185(4): 373-381.
14	Chouliaras L, Mastroeni D, Delvaux E, et al. Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer′s disease patients[J]. Neurobiol Aging, 2013, 34(9): 2091-2099.
15	Zhang XQ, Lv CJ, Liu XY, et al. Genome wide analysis of DNA methylation in rat lungs with lipopolysaccharide induced acute lung injury[J]. Mol Med Rep, 2013, 7(5): 1417-1424.
16	Lei C, Jiao Y, He B, et al. RIP140 down-regulation alleviates acute lung injury via the inhibition of LPS-induced PPARγ promoter methylation[J]. Pulm Pharmacol Ther, 2016, 37: 57-64.
17	Lin KL, Suzuki Y, Nakano H, et al. CCR2＋ monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality[J]. J Immunol, 2008, 180(4): 2562-2572.
18	Medzhitov R, Horng T. Transcriptional control of the inflammatory response[J]. Nat Rev Immunol, 2009, 9(10): 692-703.
19	Ogawa S, Lozach J, Jepsen K, et al. A nuclear receptor corepressor transcriptional checkpoint controlling activator protein 1-dependent gene networks required for macrophage activation[J]. Proc Natl Acad Sci U S A, 2004, 101(40): 14461-14466.
20	Lu R, Hu X, Zhou J, et al. COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR[J]. Nat Commun, 2016, 7: 12044.
21	Glass CK, Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells[J]. Nat Rev Immunol, 2010, 10(5): 365-376.
22	Xu F, Mao C, Ding Y, et al. Molecular and enzymatic profiles of mammalian DNA methyltransferases: structures and targets for drugs[J]. Curr Med Chem, 2010, 17(33): 4052-4071.
23	Bogdanović O, Veenstra GJ. DNA methylation and methyl-CpG binding proteins: developmental requirements and function[J]. Chromosoma, 2009, 118(5): 549-565.

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Lipopolysaccharide modulation the promoter methylation of nuclear receptor corepressor regulated inflammation mediators in macrophages

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Lipopolysaccharide modulation the promoter methylation of nuclear receptor corepressor regulated inflammation mediators in macrophages