MiR-29b-3p Inhibits LPS-induced Inflammatory Response In Bronchial Epithelial Cells By Affecting AKT2 Expression

Background and ObjectiveBronchial asthma is one of the most common chronic airway diseases in the world.Environment and heredity are the two major causes of asthma,and its pathogenesis is involved by a variety of cells.In recent years,the incidence of asthma in China has been increasing.Although glucocorticoids and ?-2 receptor agonists are commonly used drugs for the control of asthma symptoms,the side effects caused by hormone resistance and long-term use of hormones cannot be solved.MicroRNAs(miRNAs)are involved in pathophysiological processes such as airway inflammation,airway hyperresponsiveness,mucus secretion and airway remodeling,and play an important role in the pathogenesis and development of asthma.This study intends to construct an inflammatory model of bronchial epithelial cell line(BEAS-2B)by LPS in vitro,and explore the effect of miR-29b-3p on inflammatory response of bronchial epithelial cells and its possible mechanism.It is expected that this experiment will further reveal the pathogenesis of asthma and provide a new direction for the treatment of asthma.Methods1.The effects of LPS on the viability of bronchial epithelial cell line(BEAS-2B)BEAS-2B cells were treated with different concentrations of LPS(0.1?g/mL,1?g/mL,10?g/mL,100?g/mL)for 12 h,and cell viability was detected by CCK-8method.2.The effects of LPS on the production of inflammatory cytokines and the expression of miR-29b-3p in BEAS-2B cellsThe expression of inflammatory cytokines IL-6,IL-8,TNF-? and miR-29b-3p were detected by real-time fluorescent quantitative PCR after treatment of BEAS-2B cells with 1?g/mL LPS for 12 h.3.The effects of miR-29b-3p on LPS-stimulated inflammatory response of BEAS-2B cellsAfter 6 hours of starvation culture of BEAS-2B cells,miR-29b-3p mimic orinhibitor and corresponding negative control were transfected into cells,and the expression level of miR-29b-3p was detected by real-time fluorescent quantitative PCR 12 hours later.The transfected BEAS-2B cells were treated with LPS for 12 hours,and the mRNA expression levels of IL-6,IL-8 and TNF-? were detected by real-time fluorescent quantitative PCR.4.The effects of miR-29b-3p on AKT2 and LPS-induced p-AKT2 in BEAS-2B cellsAfter 6 hours of starvation culture of BEAS-2B cells,miR-29b-3p mimic or inhibitor and corresponding negative control were transfected into the cells for 12 h.The mRNA and protein expression of AKT2 were detected by real-time PCR and Western Blot.The BEAS-2B cells were treated with LPS for 12 h,and the protein expression of p-AKT2 was determined by Western Blot.5.The effects of miR-29b-3p on NF-?B signaling pathway in downstream transcription factor of AKT2After 6 hours of starvation culture of BEAS-2B cells,miR-29b-3p mimic or inhibitor and corresponding negative control were transfected into cells for 12 h,then treated with LPS for 12 h,and cytoplasmic and nuclear NF-?B p65 protein expression was determined by Western Blot.Results1.LPS affects the viability of BEAS-2B cells and can induce cellular inflammatory responses and affect the expression of miR-29b-3pCompared with the control group,different concentrations of LPS could affect the viability of BEAS-2B cells.The higher of the LPS concentration,the more obvious of the inhibitory effect.According to the above results,then treatment of BEAS-2B cells at 1?g/mL showed that the relative mRNA expression levels of IL-6,IL-8 and TNF-? were significantly increased in BEAS-2B cells after LPS stimulation compared with the control group.At the same time,the expression level of miR-29b-3p was decreased in the LPS-treated group compared with the control group.2.miR-29b-3p can inhibit LPS-induced inflammation of BEAS-2B cellsAfter transfection of miR-29b-3p mimic,the production of inflammatory cytokines TNF-?,IL-8 and IL-6 induced by LPS were significantly inhibited.In contrast,after transfection of miR-29b-3p inhibitor,the mRNA expression levels of inflammatory cytokines TNF-?,IL-6 and IL-8 were significantly up-regulated.3.miR-29b-3p affects the expression levels of AKT2 and p-AKT2 to regulate LPS-induced inflammatory responses.The mRNA and protein expression of AKT2 was decreased after transfection of miR-29b-3p mimic in BEAS-2B cells,and the mRNA and protein expression of AKT2 was increased after transfection with miR-29b-3p inhibitor.Further study of the effect of miR-29b-3p on LPS-induced p-AKT2 revealed that p-AKT2 protein expression was decreased after transfection of miR-29b-3p mimic,whereas p-AKT2 protein expression was increased transfected after miR-29b-3p inhibitor.4.miR-29b-3p can affect the nuclear translocation of NF-?B p65 in BEAS-2B cells induced by LPS.After stimulation of BEAS-2B cells by LPS,cytoplasmic NF-?B p65 decreased and NF-?B p65 increased in the nucleus,indicating that LPS activates NF-?B pathway and promotes nuclear translocation of NF-?B p65,which leads to the release of inflammatory factors.In LPS-stimulated BEAS-2B cells,nuclear translocation of NF-?B p65 was down-regulated after transfection of miR-29b-3p mimic,and nuclear translocation of NF-?B p65 was increased after transfection with miR-29b-3p inhibitor.ConclusionsThis experimental study found that miR-29b-3p was reduced in vitro by construction of LPS-induced bronchial epithelial cell inflammation model.Further studies showed that miR-29b-3p may inhibit AKT/NF-?B signaling pathway by inhibiting AKT2 expression,thereby inhibiting LPS-induced Inflammation of bronchial epithelial cells BEAS-2B.