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

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

Abstract

Abstract:

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

Xiaoli Guo, Xia Liu, Chuanjiang Lei, Guansong Wang, Jianchun Wang. Lipopolysaccharide modulation the promoter methylation of nuclear receptor corepressor regulated inflammation mediators in macrophages[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2017, 10(02): 157-162.

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URL: https://zhfbjbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-6902.2017.02.009

https://zhfbjbzz.cma-cmc.com.cn/EN/Y2017/V10/I02/157

Figures/Tables 5

References 23

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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′

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′

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