Study On The Regulation Of Human Antigen R In Airway Epithelial Cells BEAS-2B On Epithelial-mesenchymal Transition

Background:Chronic obstructive pulmonary disease(COPD)is a respiratory tract diseases with high morbidity and mortality in the worldwide.The revision of the Global Initiative for Chronic Obstructive Lung Disease(GOLD)reported in 2017 pointed out that COPD is a preventable and treatable disorder which is characterized by continual respiratory symptoms and irreversible airflow limitation.The guideline highlights that respiratory symptoms and airflow limitation are mainly caused by exposure to noxious particles or gases,no mention of chronic inflammatory response.In terms of the pathogenesis of COPD,except some consensuses include oxidative stress,protease destruction and inflammatory response,the guideline also highlights the non-inflammatory mechanisms.Non-inflammatory mechanisms contain transforming growth factor ?(TGF-?)activation,Wnt signal transduction disorder and E-cadherin decrease,which are also related with epithelial-mesenchymal transition(EMT),a process by which epithelial cells acquire a mesenchymal-like cell phenotype.Several studies have indicated that EMT is associated with tissue fibrosis,remodeling,tumorigenesis and tumor invasion and metastasis.There is evidence that EMT is closely associated with a variety of fibrotic lung diseases,including COPD.As a potential mechanism of COPD,EMT plays a role in the airway remodeling,fibrosis and even malignant and has important research value.The regulation of EMT is very complex,involving a number of signaling pathways and transcription factors.Most of the clinical targeting drug design are based on a single target,and it is difficult to achieve regulatory purposes.Human antigen R(HuR)is a widely studied RNA binding protein which can bind to a variety of messenger RNAs(mRNAs)of proteins and play different biological roles by influencing the stability or translation of mRNAs.Studies have shown that HuR is involved in the regulation of a variety of EMT related cytokines.However,there are currently no relevant reports that HuR is involved in the regulation of EMT in COPD.Objective:The purpose of this study was to clarify the expression level of HuR in airway epithelium of COPD patients,the expression and activity of HuR in human airway epithelial cells BEAS-2B induced by cigarette smoke extract(CSE)and to investigate the effect of HuR on EMT regulation at the cellular level.Methods:Immunohistochemical method was used to detect the expression of HuR in the airway epithelium of human lung tissues,and the correlation between COPD expression and the forced expiratory volume 1(FEV1)was analyzed.The experiment was divided into control group,smoking group and COPD group.Human airway epithelial cell line BEAS-2B was cultured in vitro,and cigarette smoke extract(CSE)stimulated cells to simulate COPD state.Western blot was used to detect the expression of HuR in different CSE conditions.Western blot and immunofluorescence were used to detect the expression and distribution of HuR,and according to the time of CSE stimulation,the experiment was divided into control group,3 hour group,6 hour group and the 9 hour group.Small RNA interference technique was used to inhibit the expression of HuR,and the interference efficiency was detected by real-time fluorescent quantitative PCR and Western blotting.After CSE stimulation and HuR knockdown,western blot and immunofluorescence were used to detect the expression of E-cadherin and vimentin.Light microscope was used to observe cell morphological changes.The experiment was divided into control group,CSE stimulation group and CSE stimulation +HuR interference group.After CSE stimulation and HuR interference,the expression of Zinc finger E-box-binding protein 1(ZEB-1)was detected,and the mRNA decay rate of ZEB-1 was detected after interfering HuR expression.Results:(1)HuR expression in airway epithelial cells of COPD patients was increased.Immunohistochemistry showed that the expression of HuR in COPD smoking group was significantly higher than that in control group and smoking group,and the expression of HuR in the smoking group was higher than that in the control group.There was a negative correlation between the expression level of HuR and FEV1(2)The expression and activity of HuR were increased in BEAS-2B cell line after CSE stimulation.In BEAS-2B cells,CSE stimulated the expression of HuR,and the results of Western blot and immunofluorescence showed that CSE increased the cytoplasmic distribution of HuR which mean that HuR activity is enhanced.(3)Small RNA interference can significantly reduce the expression level of HuR.BEAS-2B was transfected with 3 groups of small RNA sequences,and the expression of HuR was detected at both RNA level and protein level.The expression of HuR in 2 groups was significantly decreased.(4)HuR plays an important role in the regulation of EMT in BEAS-2B cell line stimulated by CSE.In BEAS-2B cells treated with CSE,the cells showed EMT related changes.The epithelial cell marker E-cadherin expression was decreased,the mesenchymal cell marker vimentin expression was increased and cell morphology changed into spindle shape.The expression of EMT markers and cell morphology were reversed after inhibition of HuR expression.(5)HuR mediated the regulation of ZEB-1.In BEAS-2B cells,compared with the control,the mRNA stability of ZEB-1 decreased and the degradation rate increased rapidly after HuR interference.CSE stimulated the expression of ZEB-1 and ZEB-1 expression levels was decreased after inhibiting HuR expression.Conclusion:HuR was highly expressed in airway epithelium of patients with COPD and was up-regulated in the CSE-stimulated airway epithelium BEAS-2B.In BEAS-2B,HuR was involved in the regulation of EMT and had an effect on EMT-critical transcription factor ZEB-1.