Abstract
BackgroundIncreasing evidence suggests that human antigen R (HuR) is involved in the epithelial-mesenchymal transition (EMT) process of several diseases. However, the role of HuR in EMT in the airway epithelial cells of patients with COPD remains unclear.MethodsBEAS-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.ResultsWe 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.ConclusionsEMT is partially enhanced through the HuR-binding proteins and its post-transcriptional regulation role in airway epithelium in COPD.
Highlights
Increasing evidence suggests that human antigen R (HuR) is involved in the epithelial-mesenchymal transition (EMT) process of several diseases
HuR expression was increased in airway epithelium of Chronic Obstructive Pulmonary Disease (COPD) subjects To assess HuR expression in airway epithelium of COPD subjects, lung sections from non-smoking patients without COPD patients, smokers without COPD, and smokers with COPD were stained by immunohistochemistry techniques
cigarette smoke extract (CSE) elevated HuR expression and activity in BEAS-2B cells Since cigarette smoking is the most common risk factor for COPD, BEAS-2B cells were treated with different concentrations of CSE for different time to model this microenvironment
Summary
Patients Lung tissues were obtained from 68 patients (18 nonsmoking patients without COPD, 20 smokers without COPD, and 30 smokers with COPD) at Shandong Provincial Hospital (Jinan, China). Immunohistochemical analysis was performed as previously described [10]. The primary antibodies targeted HuR (1:50) was purchased from Abcam and ZEB-1 (1:50) was purchased from Cell Signaling. Real-time PCR (RT-PCR) Total RNA was extracted from BEAS-2B cells using Trizol reagent (Invitrogen) based on manufacturer’s protocol. Western blot analysis Total cellular lysates were prepared as previously described [12]. The knockdown efficiency was tested at both mRNA and protein levels 48 h after transfection. Plasmids and transfection Human ZEB-1 expression vector was purchased from Public Protein/Plasmid Library (Nanjing, China). Immunofluorescence After the cells received their respective treatments, immunofluorescent staining was performed as previously described [13]. Samples were incubated with primary antibodies against HuR (1:100), vimentin (1:100) and E-cadherin (1:200) overnight followed by treatment with a secondary antibody labelled with Alexa Fluor 488 (Beyotime).
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