The Role Of ELAVL1/HuR In Airway Remodeling And The Corresponding Intervention Measures

BACKGROUND:Human antigen R is one of the earliest discovered RNA binding proteins.Recent studies have shown that,a variety of inflammatory factors,growth factors and cyclin mRNA molecules contains an ARE region,with the 3'UTR end,that can be recognized and regulated by HuR.Up till the present moment;,the role of HuR in many inflammatory diseases and proliferative diseases has been paid more attention due to its regulatory role in the expression of cytokine genes.In the process of chronic obstructive pulmonary disease,airway epithelial cells can produce cilia adhesion,lodging,cell vacuole degeneration,necrosis,proliferation,squamous cell epithelial metaplasia and other pathological changes.Then the abnormal pathophysiological process induce airway remodeling,airway goblet cells,mucus hypersecretion,airway obstruction,aggravating bacterial colonization and infection.In asthmatic state,smooth muscle cell cycle evolution can occur,the cells will show proliferation and hypertrophy.At the same time,smooth muscle cell extracellular matrix composition will be increased,the ultimate effect is smooth airway thickening,airway remodeling.In the phenomenon of COPD and asthma-induced abnormalities of cell morphology and function,the.regulation of mRNA stability of functional protein gene expression exists.Previously,the author and his partners have found that changes in HuR expression and activity are involved in PDGF-induced proliferation of human airway smooth muscle cells.And cyclin D1 expression is regulated by HuR.However,the role of HuR in airway remodeling under various factors is not clear.METHODS:1.OVA stimulation was used to prepare asthma models in mice,9 mouse in the experimental group and the other 9 mouse in the control group.The lung tissue of COPD patients and control group were obtained in Shandong Provincial Hospital Affiliated to Shandong University.According to the patient's medical history and smoke history,they were divided into non-smoking control group(18 cases),smoking control group(20 cases)and smoking COPD group(30 cases).Thus,immune-histochemistry was used to analyze the expression of HuR in lung tissue of mice,COPD patients and the related control groups.2.BEAS-2B cells were cultured and treated with 3%CSE.Western blotting,RT-PCR and immunofluoresence were used to detect the expression of HuR,ZEB-1.RNAi was used to suppress HuR expression.Then knockdown of HuR,RT-PCR and Western blotting showed that with siHuR-1 and siHuR-3,clear suppression of HuR expression was confirmed.We chose siHuR-3,the most effective one,to proceed with subsequent experiments.Immunofluorescence analysis,western blotting were used to detect the expression of E-cadherin,vimentin,ZEB-1 and HuR.3.We investigated the effect of PM2.5 on autophagy,and studied the effect of PM2.5-induced autophagy and AMPK on proliferation,cell cycle,apoptosis,and airway inflammation using human bronchial epithelial cells 16HBE140 cells.The effects of PM2.5 on autophagy were further studied by transmission electron microscopy and western blot.AMPK inhibitor Compound C was used to observe whether the autophagy process of airway epithelial cells could be reversed.RESULTS:1.The expression of HuR in lung tissues of COPD group and control group was observed and detected by immunohistochemical staining.The expression of HuR in COPD smoking group was significantly higher than that in control smoking group and non-smoking group(P<0.01).The cumulative optical density of each group was measured,indicating that the COPD smoking group increased significantly than the other two groups,P<0.01.Immunohistochemistry showed that HuR in OVA group was highly expressed in airway tissue.Compared with the control group,the expression of HuR was higher in asthmatic mice,and a large amount of a-SMA deposited around the blood vessels.2.The reults show that more HuR expression is enhanced in the airways epithelium of smokers with or without COPD than controls(nonsmoker non-COPD patients).However,there was no definite correlation between HuR expression and FEV1%.Further study reveals that knockdown of HuR significantly increases the apoptosis of BEAS-2B cells and down-regulates ZEB-1 expression.3.Results showed that the optimum concentration of PM2.5 treatment for cell viability inhibition was 100 ?g/ml for 24 h.PM2.5 induced cell arrest in G0/G1 phase and increased mitochondrial membrane potential with concentration.PM2.5 downregulated the Cyclin D and MMP-9 expressions but upregulated the TIMP-1 expression.PM2.5 significantly promoted IL-6 and TNF-a production.PM2.5 enhanced level and activation of AMPK.The levels of ATG5,Beclinl and LC3II/I were significantly increased by PM2.5.The activation of ULK1 was significantly inhibited by PM2.5.Moreover,ATG5 knockdown inhibited autophagy,significantly promoting Cyclin D and MMP-9 expressions but inhibiting TIMP-1 expression.PM2.5-induced IL-6 and TNF-a were significantly reversed by knockdown of ATG5.Thus,inhibition of autophagy protected cell against PM2.5-induced cell injury.Inhibition of AMPK by Compound C suppressed autophagy and protected cell against PM2.5-induced cell injury.CONCLUSIONS:1.The expression of HuR in lung tissue of COPD patients and OVA induced airway tissue in mice was increased obviously.2.EMT is partially enhanced through the HuR-binding proteins and its post-transcriptional regulation role in airway epithelium in COPD.3.PM2.5 induced human bronchial epithelial cells injury via activation of AMPK-mediated autophagy,which might provide therapeutic targets for treatment of respiratory diseases.