Effect Of MiR-155 On Human Bronchial Cell Line BEAS-2B Induced By Cigarette Smoke Extract

Background:Chronic obstructive pulmonary disease(COPD)is a global medical and economic burden,and cigarette smoke is the most important risk factor.miRNA is a relatively stable non-coding RNA in vivo,and a large number of studies have shown that miRNA is abnormally expressed during the development of COPD.Mi R-155 is a regulator of inflammatory response and has been widely studied in lung cancer,asthma and other lung diseases.SIRT1,as an important regulator of apoptosis,oxidative stress and inflammation,is involved in the development of COPD.Although current studies have shown up-regulated expression of miR-155 in body fluids of COPD patients who smoke,the overall number of studies is still small,and the specific function and mechanism of miR-155 in the development process of COPD need to be further studied.Therefore,to investigate the expression of miR-155 in human normal bronchial epithelial cells BEAS-2B induced by cigarette smoke extract(CSE)and the effect and mechanism of miR-155 inhibitors on CSE induced BEAS-2B,It provides new directions and options for elucidating the mechanism of COPD and finding markers and therapeutic targets for smoke-induced lung injury.Method:In this study,BEAS-2B cells were induced by CSE to establish the in vitro inflammation model of chronic obstructive pulmonary disease,and then the effects and mechanisms of miR-155 inhibitors on cigarette smoke-induced BEAS-2B inflammation model were explored.According to the experimental requirements,the cells were divided into the following four groups:(1)control group;(2)The CSE group was divided into 5%,10% and 15% subgroups according to the concentration of CSE.They were also divided into 0h,24 h,48h and 72 h subgroups under 10%CSE stimulation.(3)miRNA transfection groups:10%CSE+miR-155 inhibitor group and10%CSE+miR-155 NC group;(4)siRNA transfection groups:10%CSE+miR-155inhibitor+si-SIRT1 group and 10%CSE+miR-155 inhibitor+si-NC group.The proliferative activity of BEAS-2B cells in all groups was determined by CCK-8method.Real-time quantitative PCR(RT-PCR)was used to determine the expression of miR-155 in BEAS-2B cells.The contents of TNF-α,IL-6 and SIRT1 in the supernatant of BEAS-2B cells were determined by enzyme-linked immunosorbent assay(ELISA).The target genes of miR-155 action were predicted by the public database Targetscan database.The abnormal expression of miR-155 in CSE-induced BEAS-2B and the effect of miR-155 inhibitors on CSE-induced BEAS-2B inflammatory models were discussed,and the mechanism of the effect of miR-155 inhibitors on inflammatory models was further explored through silencing SIRT1.Results:The results of CCK-8 showed that the proliferative activity of BEAS-2B cells in 5%CSE group,10%CSE group and 15%CSE group was significantly decreased with the increase of concentration(p<0.05),and the proliferative activity of Beas-2B cells in 10%CSE group was not bad.After 10%CSE stimulation of BEAS-2B cells for0 h,24h,48 h and 72 h,Cell proliferation activity was always present(p<0.05).10%CSE was selected for subsequent experiments.The results of RT-PCR and ELISA showed that compared with the control group,the expressions of miR-155,IL-6 and TNF-αin inflammatory cells induced by CSE at different concentrations were increased,and the increase was more obvious with the increase of concentration(p<0.05),while the content of SIRT1 was decreased(p<0.05).After the application of miR-155 inhibitors,the above results were changed,the cell viability was increased(p<0.05),the expressions of SIRT1 was increased(p<0.05),and the expressions of IL-6 and TNF-αwas decreased(p<0.05).After silencing SIRT1 with miR-155 inhibitors,cell proliferation activity was reduced again(p<0.05),and the expressions of inflammatory cytokines IL-6 and TNF-α were increased(p<0.05).Conclusions:1.CSE can lead to the up-regulation of miR-155 expression in BEAS-2B cells.2.CSE can decrease the proliferative activity of BEAS-2B cells,decrease the inflammatory regulator SIRT1,and increase the contents of inflammatory cytokines IL-6 and TNF-α.3.miR-155 inhibitors can relieve the CSE-induced decrease in the proliferative activity of BEAS-2B cells,the decrease in the inflammatory regulator SIRT1,and the increase in the contents of inflammatory cytokines IL-6 and TNF-α;4.miR-155 is associated with SIRT1,and miR-155 inhibitors can restore the cell proliferation inhibition and the increase of inflammatory cytokines IL-6 and TNF-α caused by CSE induced BEAS-2B by regulating SIRT1.