The Effect And Mechanism Study Of EZH2 In Bronchial Epithelial Cells Inflammatory Response

Objective Bronchial asthma caused by genetic and environmental factors is a heterogeneous disease.The epithelial barrier is dysfunctional when stimulated by allergens,air pollutants and pathogenic microorganisms.This is an important mechanism of airway inflammation.Enhancer of zeste homolog 2(EZH2)can silence the target genes.EZH2 is involved in inflammation,autoimmunity and malignancies.However,few studies have reported on EZH2 in the pathogenesis of asthma.This experiment is aimed to establish a inflammatory model of BEAS-2B induced by lipopolysaccharide(LPS),and investigate into the biological role played by EZH2 and its possible mechanisms.Methods1.Constructing a inflammatory model of BEAS-2B which induced by LPS.1.1 After BEAS-2B cells were treated with LPS(0,0.1,1,10,100 μg/ml)for 12 hours,CCK-8 method was used to detect the cell viability.1.2 After BEAS-2B cells were stimulated with 10 μg/ml LPS for 12 hours,the m RNA expression of IL-6,IL-8,TNF-α,miR-139-5p and Notch1 were analysed by q RT-PCR.The protein expression of Notch1,EZH2,H3K27me3 were analysed by Western Blot.2.The effects of si-EZH2 on LPS-induced inflammatory reaction in BEAS-2B Cells.When si-EZH2 and si-NC were transfected into cells for 24 hours,the transfected cells were stimulated with 10 μg/ml LPS for another 12 hours,and the m RNA expression of IL-6,IL-8,TNF-α were analysed by q RT-PCR,and the protein expression of IL-6,IL-8,TNF-α and H3K27me3 were analysed by Western Blot.3.Analyzing the probable mechanism that si-EZH2 affects BEAS-2B cells inflammatory response induced by LPS.When si-NC,si-EZH2,mimic-NC,inhibitor-NC,miR-139-5p mimic,miR-139-5p inhibitor,si-EZH2+miR-139-5p inhibitor were transfected into cells for 24 hours,the transfected cells were stimulated with LPS for 12 hours.The m RNA expression of miR-139-5p,Notch1 were analysed by q RT-PCR,and the protein expression of Notch1 was analysed by Western Blot.Results1.LPS could induce BEAS-2B cells inflammatory response.1.1 LPS at concentrations of 0.1 μg/ml and above could affect the viability of the cells.The higher of the LPS concentration,the more significant of the inhibitory effect.We chose 10 μg/ml LPS for the following experiments.1.2 The m RNA expression of IL-6,IL-8,TNF-α,Notch1 and the protein expression of EZH2,H3K27me3,Notch1 were significantly increased,and the m RNA expression of miR-139-5p was significantly decreased after 10 μg/ml LPS treatment for 12 hours.2.Si-EZH2 could reduce LPS-induced inflammatory response in BEAS-2B cells.The cells transfected with si-EZH2 were stimulated with LPS for another 12 hours,the expression of IL-6,IL-8,TNF-α were significantly decreased.Meanwhile,the protein expression of H3K27me3 was also decreased.3.EZH2 could regulate the inflammatory reaction induced by LPS through EZH2-miR-139-5p-Notch1 axis.3.1 When si-EZH2 or miR-139-5p mimic were transfected into BEAS-2B cells,the expression of IL-6,IL-8,TNF-α were decreased.When miR-139-5p inhibitor was transfected into BEAS-2B cells,the expression of IL-6,IL-8,TNF-α were increased.si-EZH2+miR-139-5p inhibitor was able to rescue the effect of si-EZH2 on the expression of IL-6,IL-8,TNF-α.3.2 The expression of Notch1 were decreased in miR-139-5p mimic group,and increased in miR-139-5p inhibitor group.The expression of Notch1 was decreased in si-EZH2 group.si-EZH2+miR-139-5p inhibitor was able to rescue the effect of si-EZH2 on the expression of Notch1.Conclusion Silenced EZH2 could alleviate the LPS-induced BEAS-2B cells inflammatory response by upregulating the expression of miR-139-5p and downregulating the expression of target gene Notch1.