Human antigen R enhances the epithelial-mesenchymal transition via regulation of ZEB-1 in the human airway epithelium

Human antigen R enhances the epithelial-mesenchymal transition via regulation of ZEB-1 in the... Background: Increasing 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. Methods: 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. Results: We 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. Conclusions: EMT is partially enhanced through the HuR-binding proteins and its post-transcriptional regulation role in airway epithelium in COPD. Keywords: Chronic obstructive pulmonary disease, Cigarette smoke extract, Human antigen R, Epithelial–mesenchymal transition, Zinc finger E-box binding homeobox 1 Background hypersecretion, up-regulated inflammation and airway re- COPD is a disorder with a high morbidity rate and is the modeling due to repeated damage and repair of the tissue third most common cause of mortality worldwide [1]. [3]. Although many related studies have been conducted, Chronic Obstructive Pulmonary Disease (COPD) is a com- there are still few treatments that can significantly decrease mon, preventable and treatable disease that is characterized the mortality due to COPD [4]. by persistent respiratory symptoms and airflow limitation Recently, several studies have indicated that EMT, a that is due to airway and/or alveolar abnormalities usually process by which epithelial cells acquire a mesenchymal- caused by significant exposure to noxious particles or gases. like cell phenotype, is closely related to the pathogenesis Theprevalenceof COPDisdirectly related to thepreva- of COPD [5, 6]. Most of these studies investigated EMT- lence of tobacco smoking. [2]. Because of the innumerable related molecules at the transcriptional and protein levels; quantity of patients, the profound damage it causes and its however, to date, there have been few findings that were burden on society, COPD has received considerable atten- successfully applied to the clinic. We hypothesized that fo- tion. The classic pathological changes of COPD are mucus cusing on the post-transcriptional modification of mRNAs involved in EMT will be a good approach to overcome * Correspondence: docjiangshujuan@163.com this obstacle. Jian Sun and Xianmin Gu contributed equally to this work. HuR, a ubiquitously expressed RNA-binding protein Department of Respiratory Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, People’s Republic of China (RBP), is one of the best-studied members of the post- Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sun et al. Respiratory Research (2018) 19:109 Page 2 of 10 transcriptional modification family [7]. HuR selectively using an OLYMPUS IX81 light microscope (Olympus, binds to a large subset of mRNAs and influences the Tokyo, Japan) fitted with a SPOT camera. Image analysis stability and/or translation of select mRNAs which are was performed using mage-Pro Plus 6.0 software (Media Cy- implicated in different pathologies, especially cancer and bernetics, Silver Spring, MD, USA). All slides were analyzed inflammation [8]. It has been reported that HuR in a single batch by a single experienced observer with qual- mediates the EMT process in diabetic nephropathy [9]. ity assurance on randomly selected slides provided by a pro- However, the role of HuR in EMT in the airway epithe- fessional academic pathologist. lial cells of patients with COPD remains unclear. In the present study, we investigated whether HuR is involved Preparation of CSE in the cigarette smoke extract (CSE)-induced EMT CSE preparation was based on the method previously process and its corresponding mechanism. described by Aoshiba et al. [11]. Briefly, one commer- cial cigarette (Hatamen), which contains 11 mg of tar and Methods 0.8 mg of nicotine, was used in this study. A filter-free Patients cigarette was combusted using a syringe-driven instrument, Lung tissues were obtained from 68 patients (18 non- and the smoke was bubbled through 20 ml of serum-free smoking patients without COPD, 20 smokers without RPMI 1640 culture medium. The resulting suspension was COPD, and 30 smokers with COPD) at Shandong Pro- adjusted to a pH of 7.4 and filtered using a 0.22-μmpore vincial Hospital (Jinan, China). A diagnosis of COPD filter. This solution was regarded as a 100% CSE solution was based on the GOLD guidelines [2]. No subjects re- and was used within 30 min after preparation. ceived oral or inhaled corticosteroids before specimen collection. All the patients’ clinical data are shown in Cell culture Table 1. Informed consent to undergo scientific research Human bronchial epithelial (BEAS-2B) cells were ob- was obtained from all the patients before tissue collec- tained from ATCC. Cells were routinely cultured in high- tion, and the experiment was approved by the ethics glucose RPMI 1640 medium (HyClone) supplemented committee at Shandong Provincial Hospital. with 10% fetal bovine serum (Biological Industries, Israel), 100 units/ml penicillin (Invitrogen) and 100 units/ml Immunohistochemistry streptomycin (Invitrogen) and maintained at 37 °C in a HuR and ZEB-1 were immunohistochemically assessed in 100% humidified atmosphere containing 5% CO . formalin-fixed, paraffin-embedded lung tissues. Histological sections were sliced at a thickness of 4 μmand mountedon Real-time PCR (RT-PCR) poly-L-lysine-coated slides. Immunohistochemical analysis Total RNA was extracted from BEAS-2B cells using Tri- was performed as previously described [10]. The primary zol reagent (Invitrogen) based on manufacturer’s proto- antibodies targeted HuR (1:50) was purchased from Abcam col. The reverse transcription was performed according and ZEB-1 (1:50) was purchased from Cell Signaling. Color to the specification of RevertAid First Stand cDNA Syn- development was performed using a DAB color devel- thesis Kit (Thermo Scientific). RT-PCR was conducted opment kit (ZhongShan Biotech). Images were captured using SYBR Premix EX Taq (Takara), in a total reaction volume of 20 μl. The relative expression levels of target mRNAs were normalized to human β-actin expression. Table 1 Demographic characteristic of the subjects Primers sequences were shown as follows. HuR: forward, Non-smokers Smokers COPD n= 18 n=20 n=30 5’GGCGAGCATACGACA3’, reverse, 5’TATTCGGGAT Sex (female/male) 13/5 1/19 3/27 AAAGTAGC3’; β-actin: forward, 5’ AGTTGCGTTAC ACCCTTTCTTG3’, reverse, 5’ CACCTTCACCGTT Age (years) 52 ± 11 54 ± 10 58 ± 8 b e CCAGTTTT3’. Smoking history, pack-years – 26 ± 12 42 ± 25 c e FEV1 , % predicted 99 ± 12 100 ± 11 66 ± 15 Western blot analysis d e FEV1/FVC % 85 ± 7 84± 7 57± 8 Total cellular lysates were prepared as previously described GOLD stage [12]. Nuclear and cytoplasmic proteins were extracted using 1 –– 6 Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). 30 μg of each protein sample was fractionated in a 10% 2 –– 19 SDS–PAGE gels. The membranes were incubated with anti- 3 –– 5 bodies against HuR (1:5000, Abcam), E-cadherin (1:1000, 4 –– – Cell Signaling), vimentin (1:1000, Cell Signaling), ZEB-1 (1: a b COPD, chronic obstructive pulmonary disease. Pack-year, 1 year smoking 20 c d 500, Cell Signaling), HDAC1 (1:1000, Abcam) and β-actin (1: cigarettes per day. FEV1, forced expiratory volume in 1 s. FVC, forced vital capacity. Values are given as mean ± s.d 1000, ZSGB-BIO). HDAC1 was used as nuclear protein Sun et al. Respiratory Research (2018) 19:109 Page 3 of 10 internal control, and β-actin was used as cytoplasmic and As shown in Fig. 1A, more intense HuR staining was total protein internal controls. observed in the airway epithelia of smokers with or without COPD than controls (non-smoking patients Small interfering RNA (siRNA) gene silencing without COPD), especially among smokers with HuR siRNA (siHuR) and negative control siRNA (siNC) COPD. Quantitative analysis of the HuR staining (Fig. 1B) were purchased from RiboBio (Guangzhou, China). siRNA showed that HuR expression was significantly increased in constructs were transfected using the riboFECT™ CP smokers with COPD than in smokers (P < 0.01) and non- Reagent (RiboBio) according to the manufacturer’sinstruc- smokers (P < 0.01) without COPD. The HuR expression tions. The knockdown efficiency was tested at both mRNA was also higher in the smokers without COPD than in and protein levels 48 h after transfection. At 12 h after nonsmokers (P < 0.01). Then the correlation between HuR transfection, the medium was changed, and further experi- expression and FEV1% was analyzed and the results ments were conducted. siRNA sequences were as follows: showed that there was no significant correlation between siHuR-1, forward: 5′ - GGAGAACGAAUUUGAUCGU FEV1% and HuR expression (Fig. 1C-E). dTdT - 3′,reverse:3′ - dTdT CCUCUUGCUUAAAC UAGCA - 5′;siHuR-2,forward:5′ - GUCCUCGUGGAUC CSE elevated HuR expression and activity in BEAS-2B cells AGACUA dTdT - 3′,reverse:3′ -dTdT CAGGAGCACC Since cigarette smoking is the most common risk factor UAGUCUGAU - 5′;siHuR-3,forward:5′ - GGUUGCG for COPD, BEAS-2B cells were treated with different con- UUUAUCCGGUUU dTdT - 3′,reverse: 3′ - dTdT CCAA centrations of CSE for different time to model this micro- CGCAAAUAGGCCAAA - 5′. environment. Resultantly, compared with the control group (no CSE treatment), a significant elevation of HuR expres- Plasmids and transfection sion was observed after 48 h treatment of 1, 3 and 5% CSE Human ZEB-1 expression vector was purchased from Public (Fig. 2a). Of note, upon exposure to 3% CSE, the cells exhib- Protein/Plasmid Library (Nanjing, China). X-tremeGENE ited the most pronounced elevation of HuR expression. Fur- HP DNA Transfection Reagent (Roche, Indianapolis, IN, thermore, CSE was also shown to increase HuR expression USA) was used to transfect the plasmids into indicated cells. in a time-dependent manner (Fig. 2b). These findings sug- The transfection procedures followed the protocol of the gested that CSE stimulation could induce HuR expression. manufacturer. Along with its expression levels, HuR function is also associated with its subcellular distribution. More proteins Immunofluorescence will be shuttled to the cytoplasm when HuR is appropri- After the cells received their respective treatments, im- ately activated [8, 14]. We next investigated the influence munofluorescent staining was performed as previously of CSE on the subcellular distribution of HuR. Immuno- described [13]. Samples were incubated with primary fluorescence analysis illustrated that CSE stimulation for antibodies against HuR (1:100), vimentin (1:100) and 3, 6 and 9 h significantly elevated the cytoplasmatic levels E-cadherin (1:200) overnight followed by treatment with of HuR than those in control cells (Fig. 2c). Similar evi- a secondary antibody labelled with Alexa Fluor 488 dence was observed using Western blot analysis (Fig. 2d). (Beyotime). Images of the cells were captured on an At the same time, in the nuclear fraction, elevation of inverted fluorescence microscope. HuR expression was observed when the CSE treatment was prolonged from 3 h to 9 h (Fig. 2e). Taken together, Statistical analysis besides enhancing HuR expression, CSE might promote All experiments were repeated at least three times. SPSS HuR translocation from nucleus to cytoplasma as well. 17.0 software (SPSS Inc.) was used for data statistical ana- lysis. The Mann-Whitney test was applied for comparisons HuR was required for CSE-induced EMT in BEAS-2B cells between the patient groups. The Spearman test was used To test whether HuR was required for CSE-induced for correlation analyses. Student’s t-test was applied to the EMT of BEAS-2B cells, we performed targeted knock- in vitro experiments. P < 0.05 was considered statistically down of HuR using siRNA gene silencing. BEAS-2B cells significant. were transiently transfected with either one of three HuR siRNAs (siHuR-1, siHuR-2 and siHuR-3) or a nega- Results tive control siRNA (siNC) and harvested for analysis HuR expression was increased in airway epithelium of 48 h after transfection. Real-time PCR (Fig. 3a) and COPD subjects Western blot (Fig. 3b) showed that siHuR-1 and siHuR- To assess HuR expression in airway epithelium of COPD 3 clearly suppressed HuR expression. We choose siHuR-3 subjects, lung sections from non-smoking patients without (the most effective siRNA) for all subsequent experiments. COPD patients, smokers without COPD, and smokers with Western blot (Fig. 3c) and immunofluorescence analysis COPD were stained by immunohistochemistry techniques. (Fig. 3d) showed that after CSE exposure, the levels of E- Sun et al. Respiratory Research (2018) 19:109 Page 4 of 10 A Nonsmoker Smoker COPD ab c Nonsmoker B C 15 140 r = -0.06502 p > 0.05 0 60 02 46 8 Nonsmoker Smoker COPD HuR intergrated optical density Smoker COPD DE r = 0.1549 r = -0.1146 p > 0.05 p > 0.05 60 0 24 68 10 468 10 12 HuR intergrated optical density HuR intergrated optical density Fig. 1 HuR expression and its correlation with parameters of lung function in the airway epithelium. Immunohistochemical assessment of HuR expression in non-smoking subjects (n =18), smokers without COPD (n = 20), and smokers with COPD (n =30). (a) Representative HuR immunostaining (brown staining) in the airways of a nonsmoker (a), a smoker without COPD (b), and a smoker with COPD (c). (b) Quantification of HuR protein levels in the airway epithelium using the integrated optical mean density. The expression of HuR was not correlated with the predicted FEV1% in the airway epithelium of nonsmokers (c), smokers without COPD (d), and smokers with COPD (E) by Spearman’s correlation test. The values are given as the mean ± s.e.m. **P < 0.01 compared with nonsmokers and ##P < 0.01 compared with smokers without COPD cadherin (an epithelial marker) were decreased, and the that zinc finger E-box binding homeobox 1 (ZEB-1), a well- levels of vimentin (a mesenchymal marker) were in- studied transcription factor involved in carcinogenesis, plays creased. In contrast, the decrease of E-cadherin expression a pivotal role in promoting EMT and could be regulated by and the increase of vimentin expression were abolished in RNA-binding proteins. We hypothesized that HuR pro- cells treated with siHuR-3 and CSE. Moreover, the cell moted CSE-induced EMT in BEAS-2B cells by stabilizing morphological changes were also observed. As shown in ZEB-1 mRNA. Fig. 3e, after a 48 h treatment with 3% CSE, BEAS-2B cells Upon detection of the stability of ZEB-1 mRNA in exhibited fewer cell–cell contacts and had a spindle-like cells transfected with siHuR, the half-life of ZEB-1 shape compared to untreated cells. After transfected with mRNA was significantly shortened in the HuR-silenced siHuR-3, the cells restored their epithelial morphology. group versus that of the control group (Fig. 4A), suggest- These data suggested that HuR was responsible for CSE- ing that HuR might enhance ZEB-1 mRNA stability. induced EMT of BEAS-2B cells. More evidence was found at the protein level as well. Western blot analysis showed that ZEB-1 was up- HuR mediated CSE-induced EMT by stabilizing ZEB-1 regulated by CSE treatment but down-regulated after si- mRNA in BEAS-2B cells lencing HuR expression in CSE-treated cells (Fig. 4B). We proposed that some target mRNAs of HuR may be re- Immunohistochemistry analysis showed that ZEB-1 sponsible for the CSE-induced mesenchymal transformation levels were increased in smokers with COPD compared observed in BEAS-2B cells. Recent studies have indicated to those in smokers and nonsmokers without COPD, HuR intergrated optical density FEV1% in airway epithelium FEV1% FEV1% Sun et al. Respiratory Research (2018) 19:109 Page 5 of 10 Fig. 2 CSE increased HuR expression and enhanced the cytoplasmatic translocation of HuR in BEAS-2B cells. a HuR expression was examined by Western blot with various concentrations of CSE for 48 h. b HuR expression was examined by Western blot with 3% CSE for different time periods. c Immunofluorescence analysis of the subcellular distribution of HuR in BEAS-2B cells treated with 3% CSE for different time periods. The arrows refer to the cytoplasmic distribution of HuR. Western blot of HuR expression levels in the cytoplasm (d) and nucleus (e) in cells treated as described in (c) compared with the cytoplasmic marker β-actin and the nuclear marker HDAC1, respectively. Each value is presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with control group which was very similar with the expression pattern of regulated by HuR was required for CSE-induced EMT HuR(Fig. 4C and D). Statistical analysis showed that in BEAS-2B cells. ZEB-1expression was positively correlated with HuR level in airway epithelium (Fig. 4E, F and G). Functionally, over- Discussion expression of ZEB-1 in HuR-silenced BEAS-2B cells COPD is accompanied by inflammation and tissue re- resulted in an obvious decrease of E-cadherin expression modeling [15]. Tissue remodeling in COPD is character- and the increase of vimentin expression (Fig. 4H). ized by emphysema and small airway remodeling with These data indicated that ZEB-1 mRNA stabilization peribronchiolar fibrosis [16]. EMT is a process by which Sun et al. Respiratory Research (2018) 19:109 Page 6 of 10 Fig. 3 HuR was necessary for CSE-induced EMT in BEAS-2B cells. HuR mRNA (a) and protein (b) expression levels were detected using real-time PCR and Western blot analysis, respectively, with three HuR-siRNA sequences and a negative control siRNA at 48 h after transfection. Each value is presented as the mean ± SD from three independent experiments. **P < 0.01 compared with cells transfected with siNC. c, d and e) BEAS-2B cells were treated with 3% CSE for 48 h in the presence or absence of HuR gene silencing. c Western blot was used to detect the expression of E- cadherin, vimentin, and HuR. d Immunofluorescence analysis of the expression of E-cadherin and vimentin using an inversion fluorescence microscope. e Cell images were obtained using phase-contrast microscopy epithelial cells gradually lose their cell polarity and cell- markers is accompanied by reticular basement mem- cell adhesions and acquire migratory and invasive prop- brane fragmentation and reduced expression of epithelial erties similar to a mesenchymal phenotype [17]. It has junction molecules in the airways of smokers [18, 19]. been reported that EMT can cause airway remodeling/fi- Mahmood MQ et al. found that there was increased ex- brosis in COPD, as increased expression of EMT pression of EMT-related markers(EGFR, vimentin, Sun et al. Respiratory Research (2018) 19:109 Page 7 of 10 Fig. 4 (See legend on next page.) Sun et al. Respiratory Research (2018) 19:109 Page 8 of 10 (See figure on previous page.) Fig. 4 ZEB-1 was required for HuR-mediated EMT in BEAS-2B cells. (A)*P < 0.05 based on a Student’s t-test. (B) Western blot was used to detect ZEB-1 and HuR expression. BEAS-2B cells were treated with 3% CSE for 48 h in the presence or absence of HuR gene silencing. ZEB-1 expression and its correlation with HuR expression in the airway epithelium. Immunohistochemical assessment of ZEB-1 expression in non-smoking subjects (n = 18), smokers without COPD (n = 20), and smokers with COPD (n = 30). (C) Representative ZEB-1 immunostaining (brown staining) in the airways of a nonsmoker (a), a smoker without COPD (b), and a smoker with COPD (c). (D) Quantification of ZEB-1 protein levels in the airway epithelium using the integrated optical mean density. The expression of ZEB-1 was correlated with the HuR expression in the airway epithelium of nonsmokers (E), smokers without COPD (F), and smokers with COPD (G) by Spearman’s correlation test. Western blot was used to detect the expression of ZEB-1, E-cadherin and vimentin (H).The values are given as the mean ± s.e.m. **P < 0.01 compared with nonsmokers and ##P < 0.01 compared with smokers without COPD S100A4 and fragmentation) in chronic airflow limitation BEAS-2B cells after treatment with CSE in our research. small airways compared to controls. The result indicated Aside from enhancing HuR expression, our results also that EMT may be relevant to the key pathologies of incicate that CSE could promote the translocation of HuR chronic obstructive pulmonary disease, small airway fi- from nucleus to the cytoplasm. As the most prominent brosis, and airway cancers [18]. Recent investigations RNA-binding protein, HuR is predominantly localized in found that nicotine and tobacco smoke could induce the nucleus when the cell is in a quiescent state. Once EMT in BECs via the Wnt3α/β-catenin/TGF-β pathway activated, HuR could rapidly translocate from nuclear to [20]. It has been reported that the EMT biomarkers in cytoplasm, where it will exert its RNA-binding activities. airway epithelium of COPD patients varied in varying The increased cytoplasmatic HuR levels might indicate that degrees after the treatment of inhaled fluticasone propi- CSE could enhance the activities of HuR. Similar results onate (fluticasone; 500 μg twice daily for 6 months). The were also demonstrated in Michela Zago’sstudy [24]. result provided strong suggestive support for an anti- Another major finding of our present study was that EMT effect of ICS in COPD airways [21]. However, the lowering HuR expression using RNA-interference could mechanisms leading to EMT in the airways of patients effectively decrease CSE-induced EMT of BEAS-2B cells. with COPD remain largely unknown. After CSE exposure, BEAS-2B cells showed lower expression The RNA-binding protein HuR is one of the best- of E-cadherin and higher expression of vimentin, as well as studied regulators of cytoplasmic mRNAs fate. Through exhibiting a mesenchymal phenotype. Nevertheless, the post-transcriptional influence on target mRNAs, HuR can aforementioned changes were significantly reversed by HuR adjust the cellular response to inflammatory, proliferative, silencing, which indicates that HuR is required for CSE- differentiation, senescence, apoptotic, stress and immune induced EMT. Such results are in accordance with the emer- stimuli [14]. We have previously shown that altered ex- ging role of HuR in regulating the EMT process shown as in pression and activity of HuR participated in PDGF- Wan Q’sstudy [9]. induced human airway smooth muscle cell proliferation Several transcription factors, such as Snail, Slug, ZEB-1, andexpressionof cyclinD1[13]. Additionally, we demon- ZEB-2, Twist and β-catenin, have been identified as key regu- strated that an HuR/TGF-β1 feedback circuit was estab- lators of EMT and have been extensively reported [25–29]. lished to regulate airway remodeling in vivo and in vitro In Mahmood MQ’sstudy [25], β-catenin and Snail 1 expres- and that targeting this feedback loop has considerable po- sion was generally high in all subjects throughout the airway tential for treating asthma [22]. Thus, these phenomena wall with marked cytoplasmic to nuclear shift in COPD. indicate that HuR might be a significant factor which is re- Moreover, Twist expression was generalised in the epithe- sponsible for airway remodeling in asthma. But, to date, lium in normal but become more basal and nuclear with there is no report on whether HuR is implicated in COPD smoking. In our effort to elucidate the mechanism how HuR pathogenesis, especially in airway fibrosis in COPD. modulates EMT in BEAS-2B cells, we identified ZEB-1 as an In this study, we demonstrate for the first time that HuR effective mediator of these HuR-induced phenomena. It is expression altered in airway epithelium of COPD subjects. widely accepted that ZEB-1 is involved in cancer invasion in In smokers without COPD, the HuR expression levels were different tumors, including breast cancer [26], renalcellcar- higher than those in nonsmokers. Moreover, the expression cinoma [27] and esophageal squamous cancer [28]. It is of HuR in smokers with COPD was obviously higher than known that HuR silencing decreases ZEB-1 protein expres- that in the other two groups. This phenomenon indicates sion suggesting that HuR is involved in modulation of this that HuR could play a significant role in the pathogenesis gene. But, the mechanistic connection between HuR and of COPD. The pathological process is associated with ZEB-1 in CSE-induced EMT and in COPD was previously exposure to cigarette smoke. Although Hudy et al. [23] unknown. In this study, we showed that modulation of HuR reported that CSE does not induce dysregulation of the expression altered the half-life of ZEB-1 mRNA and post- RBPs AUF1 and HuR in primary human bronchial epithe- transcriptionally controlled ZEB-1 expression. Functionally, lial cells, the altered expression of HuR was validated in ZEB-1 is required for HuR-mediated EMT in BEAS-2B cells. Sun et al. Respiratory Research (2018) 19:109 Page 9 of 10 Our study reveals a mechanism by which HuR promotes Received: 1 October 2017 Accepted: 6 May 2018 CSE-induced EMT through increasing ZEB-1 expression. The previous reports showed that EMT could occur References both in large airways [30] and small airways of COPD pa- 1. Burney P, Kato B, Janson C, Mannino D, Studnicka M, Tan W, Bateman E, tients. 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Nicotine-induced epithelial-mesenchymal transition via Wnt/beta-catenin signaling in human airway epithelial cells. American journal of physiology Lung cellular and molecular physiology. 2013;304(4):L199–209. 21. Sohal SS, Soltani A, Reid D, Ward C, Wills KE, Muller HK, Walters EH. A randomized controlled trial of inhaled corticosteroids (ICS) on markers of epithelial-mesenchymal transition (EMT) in large airway samples in COPD: an exploratory proof of concept study. Int J Chron Obstruct Pulmon Dis. 2014 May 27;9:533–42. 22. Wang N, Yan D, Liu Y, Liu Y, Gu X, Sun J, Long F, Jiang S. A HuR/TGF-beta1 feedback circuit regulates airway remodeling in airway smooth muscle cells. Respir Res. 2016;17(1):117. 23. Hudy MH, Proud D. Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells. Respir Res. 2013;14:88. 24. Zago M, Sheridan JA, Nair P, Rico de Souza A, Gallouzi IE, Rousseau S, Di Marco S, Hamid Q, Eidelman DH, Baglole CJ. Aryl hydrocarbon receptor- dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding. PLoS One. 2013; 8(9):e74953. 25. Mahmood MQ, Walters EH, Shukla SD, Weston S, Muller HK, Ward C, Sohal SS. β-catenin, twist and snail: transcriptional regulation of EMT in smokers and COPD, and relation to airflow obstruction. Sci Rep. 2017;7(1):10832. 26. Preca BT, Bajdak K, Mock K, Lehmann W, Sundararajan V, Bronsert P, Matzge- Ogi A, Orian-Rousseau V, Brabletz S, Brabletz T, Maurer J, Stemmler MP. A novel ZEB1/HAS2 positive feedback loop promotes EMT in breast cancer. Oncotarget. 2017;8(7):11530–43. 27. Sun KH, Sun GH, Wu YC, Ko BJ, Hsu HT, Wu ST. TNF-alpha augments CXCR2 and CXCR3 to promote progression of renal cell carcinoma. J Cell Mol Med. 2016;20(11):2020–8. 28. 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Human antigen R enhances the epithelial-mesenchymal transition via regulation of ZEB-1 in the human airway epithelium

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Abstract

Background: Increasing 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. Methods: 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. Results: We 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. Conclusions: EMT is partially enhanced through the HuR-binding proteins and its post-transcriptional regulation role in airway epithelium in COPD. Keywords: Chronic obstructive pulmonary disease, Cigarette smoke extract, Human antigen R, Epithelial–mesenchymal transition, Zinc finger E-box binding homeobox 1 Background hypersecretion, up-regulated inflammation and airway re- COPD is a disorder with a high morbidity rate and is the modeling due to repeated damage and repair of the tissue third most common cause of mortality worldwide [1]. [3]. Although many related studies have been conducted, Chronic Obstructive Pulmonary Disease (COPD) is a com- there are still few treatments that can significantly decrease mon, preventable and treatable disease that is characterized the mortality due to COPD [4]. by persistent respiratory symptoms and airflow limitation Recently, several studies have indicated that EMT, a that is due to airway and/or alveolar abnormalities usually process by which epithelial cells acquire a mesenchymal- caused by significant exposure to noxious particles or gases. like cell phenotype, is closely related to the pathogenesis Theprevalenceof COPDisdirectly related to thepreva- of COPD [5, 6]. Most of these studies investigated EMT- lence of tobacco smoking. [2]. Because of the innumerable related molecules at the transcriptional and protein levels; quantity of patients, the profound damage it causes and its however, to date, there have been few findings that were burden on society, COPD has received considerable atten- successfully applied to the clinic. We hypothesized that fo- tion. The classic pathological changes of COPD are mucus cusing on the post-transcriptional modification of mRNAs involved in EMT will be a good approach to overcome * Correspondence: docjiangshujuan@163.com this obstacle. Jian Sun and Xianmin Gu contributed equally to this work. HuR, a ubiquitously expressed RNA-binding protein Department of Respiratory Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, People’s Republic of China (RBP), is one of the best-studied members of the post- Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sun et al. Respiratory Research (2018) 19:109 Page 2 of 10 transcriptional modification family [7]. HuR selectively using an OLYMPUS IX81 light microscope (Olympus, binds to a large subset of mRNAs and influences the Tokyo, Japan) fitted with a SPOT camera. Image analysis stability and/or translation of select mRNAs which are was performed using mage-Pro Plus 6.0 software (Media Cy- implicated in different pathologies, especially cancer and bernetics, Silver Spring, MD, USA). All slides were analyzed inflammation [8]. It has been reported that HuR in a single batch by a single experienced observer with qual- mediates the EMT process in diabetic nephropathy [9]. ity assurance on randomly selected slides provided by a pro- However, the role of HuR in EMT in the airway epithe- fessional academic pathologist. lial cells of patients with COPD remains unclear. In the present study, we investigated whether HuR is involved Preparation of CSE in the cigarette smoke extract (CSE)-induced EMT CSE preparation was based on the method previously process and its corresponding mechanism. described by Aoshiba et al. [11]. Briefly, one commer- cial cigarette (Hatamen), which contains 11 mg of tar and Methods 0.8 mg of nicotine, was used in this study. A filter-free Patients cigarette was combusted using a syringe-driven instrument, Lung tissues were obtained from 68 patients (18 non- and the smoke was bubbled through 20 ml of serum-free smoking patients without COPD, 20 smokers without RPMI 1640 culture medium. The resulting suspension was COPD, and 30 smokers with COPD) at Shandong Pro- adjusted to a pH of 7.4 and filtered using a 0.22-μmpore vincial Hospital (Jinan, China). A diagnosis of COPD filter. This solution was regarded as a 100% CSE solution was based on the GOLD guidelines [2]. No subjects re- and was used within 30 min after preparation. ceived oral or inhaled corticosteroids before specimen collection. All the patients’ clinical data are shown in Cell culture Table 1. Informed consent to undergo scientific research Human bronchial epithelial (BEAS-2B) cells were ob- was obtained from all the patients before tissue collec- tained from ATCC. Cells were routinely cultured in high- tion, and the experiment was approved by the ethics glucose RPMI 1640 medium (HyClone) supplemented committee at Shandong Provincial Hospital. with 10% fetal bovine serum (Biological Industries, Israel), 100 units/ml penicillin (Invitrogen) and 100 units/ml Immunohistochemistry streptomycin (Invitrogen) and maintained at 37 °C in a HuR and ZEB-1 were immunohistochemically assessed in 100% humidified atmosphere containing 5% CO . formalin-fixed, paraffin-embedded lung tissues. Histological sections were sliced at a thickness of 4 μmand mountedon Real-time PCR (RT-PCR) poly-L-lysine-coated slides. Immunohistochemical analysis Total RNA was extracted from BEAS-2B cells using Tri- was performed as previously described [10]. The primary zol reagent (Invitrogen) based on manufacturer’s proto- antibodies targeted HuR (1:50) was purchased from Abcam col. The reverse transcription was performed according and ZEB-1 (1:50) was purchased from Cell Signaling. Color to the specification of RevertAid First Stand cDNA Syn- development was performed using a DAB color devel- thesis Kit (Thermo Scientific). RT-PCR was conducted opment kit (ZhongShan Biotech). Images were captured using SYBR Premix EX Taq (Takara), in a total reaction volume of 20 μl. The relative expression levels of target mRNAs were normalized to human β-actin expression. Table 1 Demographic characteristic of the subjects Primers sequences were shown as follows. HuR: forward, Non-smokers Smokers COPD n= 18 n=20 n=30 5’GGCGAGCATACGACA3’, reverse, 5’TATTCGGGAT Sex (female/male) 13/5 1/19 3/27 AAAGTAGC3’; β-actin: forward, 5’ AGTTGCGTTAC ACCCTTTCTTG3’, reverse, 5’ CACCTTCACCGTT Age (years) 52 ± 11 54 ± 10 58 ± 8 b e CCAGTTTT3’. Smoking history, pack-years – 26 ± 12 42 ± 25 c e FEV1 , % predicted 99 ± 12 100 ± 11 66 ± 15 Western blot analysis d e FEV1/FVC % 85 ± 7 84± 7 57± 8 Total cellular lysates were prepared as previously described GOLD stage [12]. Nuclear and cytoplasmic proteins were extracted using 1 –– 6 Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). 30 μg of each protein sample was fractionated in a 10% 2 –– 19 SDS–PAGE gels. The membranes were incubated with anti- 3 –– 5 bodies against HuR (1:5000, Abcam), E-cadherin (1:1000, 4 –– – Cell Signaling), vimentin (1:1000, Cell Signaling), ZEB-1 (1: a b COPD, chronic obstructive pulmonary disease. Pack-year, 1 year smoking 20 c d 500, Cell Signaling), HDAC1 (1:1000, Abcam) and β-actin (1: cigarettes per day. FEV1, forced expiratory volume in 1 s. FVC, forced vital capacity. Values are given as mean ± s.d 1000, ZSGB-BIO). HDAC1 was used as nuclear protein Sun et al. Respiratory Research (2018) 19:109 Page 3 of 10 internal control, and β-actin was used as cytoplasmic and As shown in Fig. 1A, more intense HuR staining was total protein internal controls. observed in the airway epithelia of smokers with or without COPD than controls (non-smoking patients Small interfering RNA (siRNA) gene silencing without COPD), especially among smokers with HuR siRNA (siHuR) and negative control siRNA (siNC) COPD. Quantitative analysis of the HuR staining (Fig. 1B) were purchased from RiboBio (Guangzhou, China). siRNA showed that HuR expression was significantly increased in constructs were transfected using the riboFECT™ CP smokers with COPD than in smokers (P < 0.01) and non- Reagent (RiboBio) according to the manufacturer’sinstruc- smokers (P < 0.01) without COPD. The HuR expression tions. The knockdown efficiency was tested at both mRNA was also higher in the smokers without COPD than in and protein levels 48 h after transfection. At 12 h after nonsmokers (P < 0.01). Then the correlation between HuR transfection, the medium was changed, and further experi- expression and FEV1% was analyzed and the results ments were conducted. siRNA sequences were as follows: showed that there was no significant correlation between siHuR-1, forward: 5′ - GGAGAACGAAUUUGAUCGU FEV1% and HuR expression (Fig. 1C-E). dTdT - 3′,reverse:3′ - dTdT CCUCUUGCUUAAAC UAGCA - 5′;siHuR-2,forward:5′ - GUCCUCGUGGAUC CSE elevated HuR expression and activity in BEAS-2B cells AGACUA dTdT - 3′,reverse:3′ -dTdT CAGGAGCACC Since cigarette smoking is the most common risk factor UAGUCUGAU - 5′;siHuR-3,forward:5′ - GGUUGCG for COPD, BEAS-2B cells were treated with different con- UUUAUCCGGUUU dTdT - 3′,reverse: 3′ - dTdT CCAA centrations of CSE for different time to model this micro- CGCAAAUAGGCCAAA - 5′. environment. Resultantly, compared with the control group (no CSE treatment), a significant elevation of HuR expres- Plasmids and transfection sion was observed after 48 h treatment of 1, 3 and 5% CSE Human ZEB-1 expression vector was purchased from Public (Fig. 2a). Of note, upon exposure to 3% CSE, the cells exhib- Protein/Plasmid Library (Nanjing, China). X-tremeGENE ited the most pronounced elevation of HuR expression. Fur- HP DNA Transfection Reagent (Roche, Indianapolis, IN, thermore, CSE was also shown to increase HuR expression USA) was used to transfect the plasmids into indicated cells. in a time-dependent manner (Fig. 2b). These findings sug- The transfection procedures followed the protocol of the gested that CSE stimulation could induce HuR expression. manufacturer. Along with its expression levels, HuR function is also associated with its subcellular distribution. More proteins Immunofluorescence will be shuttled to the cytoplasm when HuR is appropri- After the cells received their respective treatments, im- ately activated [8, 14]. We next investigated the influence munofluorescent staining was performed as previously of CSE on the subcellular distribution of HuR. Immuno- described [13]. Samples were incubated with primary fluorescence analysis illustrated that CSE stimulation for antibodies against HuR (1:100), vimentin (1:100) and 3, 6 and 9 h significantly elevated the cytoplasmatic levels E-cadherin (1:200) overnight followed by treatment with of HuR than those in control cells (Fig. 2c). Similar evi- a secondary antibody labelled with Alexa Fluor 488 dence was observed using Western blot analysis (Fig. 2d). (Beyotime). Images of the cells were captured on an At the same time, in the nuclear fraction, elevation of inverted fluorescence microscope. HuR expression was observed when the CSE treatment was prolonged from 3 h to 9 h (Fig. 2e). Taken together, Statistical analysis besides enhancing HuR expression, CSE might promote All experiments were repeated at least three times. SPSS HuR translocation from nucleus to cytoplasma as well. 17.0 software (SPSS Inc.) was used for data statistical ana- lysis. The Mann-Whitney test was applied for comparisons HuR was required for CSE-induced EMT in BEAS-2B cells between the patient groups. The Spearman test was used To test whether HuR was required for CSE-induced for correlation analyses. Student’s t-test was applied to the EMT of BEAS-2B cells, we performed targeted knock- in vitro experiments. P < 0.05 was considered statistically down of HuR using siRNA gene silencing. BEAS-2B cells significant. were transiently transfected with either one of three HuR siRNAs (siHuR-1, siHuR-2 and siHuR-3) or a nega- Results tive control siRNA (siNC) and harvested for analysis HuR expression was increased in airway epithelium of 48 h after transfection. Real-time PCR (Fig. 3a) and COPD subjects Western blot (Fig. 3b) showed that siHuR-1 and siHuR- To assess HuR expression in airway epithelium of COPD 3 clearly suppressed HuR expression. We choose siHuR-3 subjects, lung sections from non-smoking patients without (the most effective siRNA) for all subsequent experiments. COPD patients, smokers without COPD, and smokers with Western blot (Fig. 3c) and immunofluorescence analysis COPD were stained by immunohistochemistry techniques. (Fig. 3d) showed that after CSE exposure, the levels of E- Sun et al. Respiratory Research (2018) 19:109 Page 4 of 10 A Nonsmoker Smoker COPD ab c Nonsmoker B C 15 140 r = -0.06502 p > 0.05 0 60 02 46 8 Nonsmoker Smoker COPD HuR intergrated optical density Smoker COPD DE r = 0.1549 r = -0.1146 p > 0.05 p > 0.05 60 0 24 68 10 468 10 12 HuR intergrated optical density HuR intergrated optical density Fig. 1 HuR expression and its correlation with parameters of lung function in the airway epithelium. Immunohistochemical assessment of HuR expression in non-smoking subjects (n =18), smokers without COPD (n = 20), and smokers with COPD (n =30). (a) Representative HuR immunostaining (brown staining) in the airways of a nonsmoker (a), a smoker without COPD (b), and a smoker with COPD (c). (b) Quantification of HuR protein levels in the airway epithelium using the integrated optical mean density. The expression of HuR was not correlated with the predicted FEV1% in the airway epithelium of nonsmokers (c), smokers without COPD (d), and smokers with COPD (E) by Spearman’s correlation test. The values are given as the mean ± s.e.m. **P < 0.01 compared with nonsmokers and ##P < 0.01 compared with smokers without COPD cadherin (an epithelial marker) were decreased, and the that zinc finger E-box binding homeobox 1 (ZEB-1), a well- levels of vimentin (a mesenchymal marker) were in- studied transcription factor involved in carcinogenesis, plays creased. In contrast, the decrease of E-cadherin expression a pivotal role in promoting EMT and could be regulated by and the increase of vimentin expression were abolished in RNA-binding proteins. We hypothesized that HuR pro- cells treated with siHuR-3 and CSE. Moreover, the cell moted CSE-induced EMT in BEAS-2B cells by stabilizing morphological changes were also observed. As shown in ZEB-1 mRNA. Fig. 3e, after a 48 h treatment with 3% CSE, BEAS-2B cells Upon detection of the stability of ZEB-1 mRNA in exhibited fewer cell–cell contacts and had a spindle-like cells transfected with siHuR, the half-life of ZEB-1 shape compared to untreated cells. After transfected with mRNA was significantly shortened in the HuR-silenced siHuR-3, the cells restored their epithelial morphology. group versus that of the control group (Fig. 4A), suggest- These data suggested that HuR was responsible for CSE- ing that HuR might enhance ZEB-1 mRNA stability. induced EMT of BEAS-2B cells. More evidence was found at the protein level as well. Western blot analysis showed that ZEB-1 was up- HuR mediated CSE-induced EMT by stabilizing ZEB-1 regulated by CSE treatment but down-regulated after si- mRNA in BEAS-2B cells lencing HuR expression in CSE-treated cells (Fig. 4B). We proposed that some target mRNAs of HuR may be re- Immunohistochemistry analysis showed that ZEB-1 sponsible for the CSE-induced mesenchymal transformation levels were increased in smokers with COPD compared observed in BEAS-2B cells. Recent studies have indicated to those in smokers and nonsmokers without COPD, HuR intergrated optical density FEV1% in airway epithelium FEV1% FEV1% Sun et al. Respiratory Research (2018) 19:109 Page 5 of 10 Fig. 2 CSE increased HuR expression and enhanced the cytoplasmatic translocation of HuR in BEAS-2B cells. a HuR expression was examined by Western blot with various concentrations of CSE for 48 h. b HuR expression was examined by Western blot with 3% CSE for different time periods. c Immunofluorescence analysis of the subcellular distribution of HuR in BEAS-2B cells treated with 3% CSE for different time periods. The arrows refer to the cytoplasmic distribution of HuR. Western blot of HuR expression levels in the cytoplasm (d) and nucleus (e) in cells treated as described in (c) compared with the cytoplasmic marker β-actin and the nuclear marker HDAC1, respectively. Each value is presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with control group which was very similar with the expression pattern of regulated by HuR was required for CSE-induced EMT HuR(Fig. 4C and D). Statistical analysis showed that in BEAS-2B cells. ZEB-1expression was positively correlated with HuR level in airway epithelium (Fig. 4E, F and G). Functionally, over- Discussion expression of ZEB-1 in HuR-silenced BEAS-2B cells COPD is accompanied by inflammation and tissue re- resulted in an obvious decrease of E-cadherin expression modeling [15]. Tissue remodeling in COPD is character- and the increase of vimentin expression (Fig. 4H). ized by emphysema and small airway remodeling with These data indicated that ZEB-1 mRNA stabilization peribronchiolar fibrosis [16]. EMT is a process by which Sun et al. Respiratory Research (2018) 19:109 Page 6 of 10 Fig. 3 HuR was necessary for CSE-induced EMT in BEAS-2B cells. HuR mRNA (a) and protein (b) expression levels were detected using real-time PCR and Western blot analysis, respectively, with three HuR-siRNA sequences and a negative control siRNA at 48 h after transfection. Each value is presented as the mean ± SD from three independent experiments. **P < 0.01 compared with cells transfected with siNC. c, d and e) BEAS-2B cells were treated with 3% CSE for 48 h in the presence or absence of HuR gene silencing. c Western blot was used to detect the expression of E- cadherin, vimentin, and HuR. d Immunofluorescence analysis of the expression of E-cadherin and vimentin using an inversion fluorescence microscope. e Cell images were obtained using phase-contrast microscopy epithelial cells gradually lose their cell polarity and cell- markers is accompanied by reticular basement mem- cell adhesions and acquire migratory and invasive prop- brane fragmentation and reduced expression of epithelial erties similar to a mesenchymal phenotype [17]. It has junction molecules in the airways of smokers [18, 19]. been reported that EMT can cause airway remodeling/fi- Mahmood MQ et al. found that there was increased ex- brosis in COPD, as increased expression of EMT pression of EMT-related markers(EGFR, vimentin, Sun et al. Respiratory Research (2018) 19:109 Page 7 of 10 Fig. 4 (See legend on next page.) Sun et al. Respiratory Research (2018) 19:109 Page 8 of 10 (See figure on previous page.) Fig. 4 ZEB-1 was required for HuR-mediated EMT in BEAS-2B cells. (A)*P < 0.05 based on a Student’s t-test. (B) Western blot was used to detect ZEB-1 and HuR expression. BEAS-2B cells were treated with 3% CSE for 48 h in the presence or absence of HuR gene silencing. ZEB-1 expression and its correlation with HuR expression in the airway epithelium. Immunohistochemical assessment of ZEB-1 expression in non-smoking subjects (n = 18), smokers without COPD (n = 20), and smokers with COPD (n = 30). (C) Representative ZEB-1 immunostaining (brown staining) in the airways of a nonsmoker (a), a smoker without COPD (b), and a smoker with COPD (c). (D) Quantification of ZEB-1 protein levels in the airway epithelium using the integrated optical mean density. The expression of ZEB-1 was correlated with the HuR expression in the airway epithelium of nonsmokers (E), smokers without COPD (F), and smokers with COPD (G) by Spearman’s correlation test. Western blot was used to detect the expression of ZEB-1, E-cadherin and vimentin (H).The values are given as the mean ± s.e.m. **P < 0.01 compared with nonsmokers and ##P < 0.01 compared with smokers without COPD S100A4 and fragmentation) in chronic airflow limitation BEAS-2B cells after treatment with CSE in our research. small airways compared to controls. The result indicated Aside from enhancing HuR expression, our results also that EMT may be relevant to the key pathologies of incicate that CSE could promote the translocation of HuR chronic obstructive pulmonary disease, small airway fi- from nucleus to the cytoplasm. As the most prominent brosis, and airway cancers [18]. Recent investigations RNA-binding protein, HuR is predominantly localized in found that nicotine and tobacco smoke could induce the nucleus when the cell is in a quiescent state. Once EMT in BECs via the Wnt3α/β-catenin/TGF-β pathway activated, HuR could rapidly translocate from nuclear to [20]. It has been reported that the EMT biomarkers in cytoplasm, where it will exert its RNA-binding activities. airway epithelium of COPD patients varied in varying The increased cytoplasmatic HuR levels might indicate that degrees after the treatment of inhaled fluticasone propi- CSE could enhance the activities of HuR. Similar results onate (fluticasone; 500 μg twice daily for 6 months). The were also demonstrated in Michela Zago’sstudy [24]. result provided strong suggestive support for an anti- Another major finding of our present study was that EMT effect of ICS in COPD airways [21]. However, the lowering HuR expression using RNA-interference could mechanisms leading to EMT in the airways of patients effectively decrease CSE-induced EMT of BEAS-2B cells. with COPD remain largely unknown. After CSE exposure, BEAS-2B cells showed lower expression The RNA-binding protein HuR is one of the best- of E-cadherin and higher expression of vimentin, as well as studied regulators of cytoplasmic mRNAs fate. Through exhibiting a mesenchymal phenotype. Nevertheless, the post-transcriptional influence on target mRNAs, HuR can aforementioned changes were significantly reversed by HuR adjust the cellular response to inflammatory, proliferative, silencing, which indicates that HuR is required for CSE- differentiation, senescence, apoptotic, stress and immune induced EMT. Such results are in accordance with the emer- stimuli [14]. We have previously shown that altered ex- ging role of HuR in regulating the EMT process shown as in pression and activity of HuR participated in PDGF- Wan Q’sstudy [9]. induced human airway smooth muscle cell proliferation Several transcription factors, such as Snail, Slug, ZEB-1, andexpressionof cyclinD1[13]. Additionally, we demon- ZEB-2, Twist and β-catenin, have been identified as key regu- strated that an HuR/TGF-β1 feedback circuit was estab- lators of EMT and have been extensively reported [25–29]. lished to regulate airway remodeling in vivo and in vitro In Mahmood MQ’sstudy [25], β-catenin and Snail 1 expres- and that targeting this feedback loop has considerable po- sion was generally high in all subjects throughout the airway tential for treating asthma [22]. Thus, these phenomena wall with marked cytoplasmic to nuclear shift in COPD. indicate that HuR might be a significant factor which is re- Moreover, Twist expression was generalised in the epithe- sponsible for airway remodeling in asthma. But, to date, lium in normal but become more basal and nuclear with there is no report on whether HuR is implicated in COPD smoking. In our effort to elucidate the mechanism how HuR pathogenesis, especially in airway fibrosis in COPD. modulates EMT in BEAS-2B cells, we identified ZEB-1 as an In this study, we demonstrate for the first time that HuR effective mediator of these HuR-induced phenomena. It is expression altered in airway epithelium of COPD subjects. widely accepted that ZEB-1 is involved in cancer invasion in In smokers without COPD, the HuR expression levels were different tumors, including breast cancer [26], renalcellcar- higher than those in nonsmokers. Moreover, the expression cinoma [27] and esophageal squamous cancer [28]. It is of HuR in smokers with COPD was obviously higher than known that HuR silencing decreases ZEB-1 protein expres- that in the other two groups. This phenomenon indicates sion suggesting that HuR is involved in modulation of this that HuR could play a significant role in the pathogenesis gene. But, the mechanistic connection between HuR and of COPD. The pathological process is associated with ZEB-1 in CSE-induced EMT and in COPD was previously exposure to cigarette smoke. Although Hudy et al. [23] unknown. In this study, we showed that modulation of HuR reported that CSE does not induce dysregulation of the expression altered the half-life of ZEB-1 mRNA and post- RBPs AUF1 and HuR in primary human bronchial epithe- transcriptionally controlled ZEB-1 expression. Functionally, lial cells, the altered expression of HuR was validated in ZEB-1 is required for HuR-mediated EMT in BEAS-2B cells. Sun et al. Respiratory Research (2018) 19:109 Page 9 of 10 Our study reveals a mechanism by which HuR promotes Received: 1 October 2017 Accepted: 6 May 2018 CSE-induced EMT through increasing ZEB-1 expression. The previous reports showed that EMT could occur References both in large airways [30] and small airways of COPD pa- 1. Burney P, Kato B, Janson C, Mannino D, Studnicka M, Tan W, Bateman E, tients. In this study, we focus on the EMT process in small Koçabas A, Vollmer WM, Gislason T, Marks G, Koul PA, Gnatiuc L, Buist S. airways (2.5 mm internal diameter). Actually, we didn’t Burden of obstructive lung disease (BOLD) study. Chronic obstructive pulmonary disease mortality and prevalence: the associations with smoking observe the HuR and ZEB-1 expression in large airways. and poverty–a BOLD analysis. Thorax. 2014;69(5):465–73. We think that whether HuR and ZEB-1 play the same role 2. Vogelmeier CF, Criner GJ, Martinez FJ, Anzueto A, Barnes PJ, Bourbeau J, in large airways will be the next step of our research. Celli BR, Chen R, Decramer M, Fabbri LM, Frith P, Halpin DM, López Varela MV, Nishimura M, Roche N, Rodriguez-Roisin R, Sin DD, Singh D, Stockley R, Vestbo J, Wedzicha JA, Agustí A. Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 Conclusion report. GOLD executive summary. 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Ohashi S, Natsuizaka M, Naganuma S, Kagawa S, Kimura S, Itoh H, Kalman RA, Nakagawa M, Darling DS, Basu D, Gimotty PA, Klein-Szanto AJ, Diehl JA, Herlyn M, Nakagawa H. A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors. Cancer Res. 2011;71(21):6836–47. 29. Mahmood MQ, Reid D, Ward C, Muller HK, Knight DA, Sohal SS, Walters EH. Transforming growth factor (TGF) β1 and Smad signalling pathways: a likely key to EMT-associated COPD pathogenesis. Respirology. 2017;22(1):133–40. 30. Sohal SS, Reid D, Soltani A, Ward C, Weston S, Muller HK, Wood-Baker R, Walters EH. Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease. Respirology. 2010;15(6):930–8.

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Respiratory ResearchSpringer Journals

Published: Jun 4, 2018

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