LncRNA-ATB Regulates EMT By Competitively Sponging MiR-200c To Promote Silica-induced Pulmonary Fibrosis

Silicosis is a pulmonary disease characterized by irreversible lung fibrosis.It's the most common,dangerous and fastest developing type of pneumoconiosis.As an occupational disease,silicosis caused heavy burden in developing countries,also,seriously threatened the life quality of workers.The inhaled silica dust enters the lung and stimulates different types of effector cells during the process of production.The initial stage is mainly inflammation,and transfers to fibrosis stage gradually.The occurrence of silicosis involves different stages,many types of cells,a large number of inflammatory cytokines,fibrogenic factors,related genes,and regulatory factors,together,they form a complex regulatory network.The pathogenesis of silicosis is complicated and has not been fully understood,which restricts us to find the effective method of prevention and treatment.Therefore,it's with great economic and social value to further study the molecular mechanism of silicosis.Epithelial-mesenchymal transition?EMT?plays an important role in many diseases.During the process,the polarity of the epithelial cells disappeared and loses the connection with the basement membrane,thereby gaining the ability of metastasis and invasion.Also,the cytoskeleton is reorganized,and the cell morphology becomes spindle-shaped.In the meanwhile,the expression of E-cadherin is decreased,and the characteristics of fibroblasts or mesenchymal cells are prominent.For example,the protein levels of Vimentin,N-cadherin,and Snail would increase,as well as secreted pro-fibrogenic factors including Collagen I and CTGF.The EMT process of lung epithelial cells is a key biology event during pulmonary fibrosis.The transformed cells promote lung fibrosis by activating the fibroblasts/myofibroblasts and accelerating the deposition of collagen.However,the detailed molecular mechanism of EMT process during pulmonary fibrosis is not fully understood.Long non-coding RNA?lncRNAs?is a kind of long RNA transcript without the protein coding ability,it widely involves in various aspects of cellular processes.Former studies suggest that the abnormal expression of lncRNA is closely related to the development and prognosis of many human diseases.Hence,dysregulated lncRNA may function as the important biological marker for various diseases including pulmonary fibrosis.LncRNAs play their roles in different ways like modification of transcription,translation and post-translational levels.Recently,lncRNA has attracted extensive attention as a competitive endogenous RNA?ceRNA?that binds to microRNA?miRNA?through a“sponge”adsorption mechanism.Which means,in addition to miRNA promoted mRNA degradation and post-transcriptional translation inhibition,lncRNAs could reversely regulate miRNAs by competing with mRNAs for binding to miRNAs.At present,the role of lncRNA as ceRNA in tumor has been reported broadly,however,only few researches reported similar role of lncRNA in idiopathic pulmonary fibrosis?IPF?.Interestingly,the researchers from China detected expression levels of lncRNA-ATB in plasma of coal workers'pneumoconiosis patients.The results suggested the lncRNA-ATB significantly increased in patients,and were positively correlated with the levels of TGF-?1.Our study also suggested elevated expression of lncRNA-ATB in TGF-?1 treated lung epithelium,companied with EMT process.Further knockdown of lncRNA-ATB significantly blocked TGF-?1-induced EMT.To verify the potential ceRNA role of lncRNA-ATB played during EMT,we performed miRNA array by lncRNA-ATB knockdown Beas-2B cells.Through qRT-PCR verification and bioformatics analysis,we found miR-200c was the most significantly affected miRNA by lnc RNA-ATB,also has one potential binding site with lncRNA-ATB.The detailed molecular mechanism of lncRNA-ATB was not clear in pulmonary fibrosis.Hence,our research mainly focused on how lncRNA-ATB involved in epithelium EMT during silica-induced lung fibrosis,trying to figure out the miRNA bound with lncRNA-ATB as ceRNA.The study will provide strong scientific evidence for early detected biomarkers and therapy targets of silicosis.ObjectiveWe aimed to study how lncRNA-ATB functions as miRNA sponge,trying to figure out the underlying regulatory mechnism and related signal molecules in vitro.We also investigated the role of lncRNA-ATB sponged miRNA during silica-induced pulmonary fibrosis in vivo.Further revealing the molecular mechanism of non-coding RNA regulated EMT process in silicosis.MethodsWe established TGF-?1 induced EMT cell models by using human alveolar type II epithelial A549 cells and human bronchial epithelial Beas-2B cells.Wound-healing assay was performed to verify migration ability of cells.Western Blot and Immunostaining were used to examine expression levels of EMT related protein markers.We also localized the lncRNA-ATB in cells by isolation of cytoplasmic and nuclear RNA.LncRNA-ATB siRNAs were constructed and transfected into Beas-2B cells to observe the regulatory role of lncRNA-ATB in EMT process.Affymetrix GeneChip miRNA 4.0 array screened lncRNA-ATB influenced potential miRNAs,verified by qRT-PCR.RT-PCR together with qRT-PCR were performed to detect levels of lncRNA-ATB.To prove the binding between miR-200c and lncRNA-ATB,dual luciferase reporter gene assay and RNA pull-down were used.We co-transfected lncRNA-ATB siRNA and miR-200c mimic to observe their combined inhibitory effect on target gene ZEB1 and EMT.LncRNA-ATB siRNA together with miR-200c inhibitor or lncRNA-ATB plasmid with miR-200c mimic were used to perform function rescue experiments,which is helpful to explain their relationship.Further,we established silica-induced lung fibrosis model and miR-200c agomir model via tail vein by C57BL/6 mice.And we observed the pathologic change of mice lungs to evaluate the degree of fibrosis.Western Blot were used to detect fibrosis and EMT markers,Immunohistochemistry assay were used to examine the expression levels of Collogen I,and hydroxyproline content was also measured.To figure out the resource of elevated level of lncRNA-ATB in lung epithelial cells,we performed co-culture experiment.PMA was used to induce THP-1 differentiate into macrophages.Next,we stimulated the macrophages by silica.Fluorescence-activated cell sorting?FACS?analysis was used to sort M2 type macrophages and co-cultured with Beas-2B cells,then,we observed the changes of cell morphologic and EMT markers,detected expression levels of lncRNA-ATB and miR-200c in Beas-2B.Results1.Elevated expression of lncRNA-ATB involves in EMT process of lung epitheliumFirstly,we constructed EMT models in A549 and Beas-2B cells by using 0,1,2,5 ng/mL TGF-?1 for 24h or 48h.According to cell morphology,migration ability,and EMT markers,we found 5 ng ng/m L TGF-?1 treated for 48h induced most significantly change,which is used in the subsequent EMT models.Subsequently,we measured expression levels of lncRNA-ATB in this model,the levels of lncRNA-ATB elevated gradually with higher dose of TGF-?1 and longer treatment.These results indicated lncRNA-ATB may involve in EMT process of lung epithelial cells.Then,we treated TGF-?1 stimulated Beas-2B cells with lncRNA-ATB siRNA,which successfully knockdown lncRNA-ATB.Compared with EMT model cells,knockdown of lncRNA-ATB elevated expression of E-cadherin,while mesenchymal and fibrosis markers including Vimentin,ZEB1,Fibronectin,and?-SMA levels were down-regulated.These results suggested knockdown of lncRNA-ATB relieved TGF-?1 induced EMT,proved regulatory role of lncRNA-ATB during this process in lung epithelium.2.LncRNA-ATB directly binding with miR-200c during EMTTo confirm the molecular mechanism of lncRNA-ATB regulated EMT,we performed isolation of cytoplasmic and nuclear RNA assay.The result showed that lncRNA-ATB was enriched in cytoplasm instead of nuclear,which is a characteristic of ceRNA function for lncRNA.So we mainly focused on its ceRNA role in the following study.Next,miRNA miRNA microarray was accomplished with TGF-?1treated Beas-2B cells,and TGF-?1+lncRNA-ATB siRNA treated Beas-2B cells.We verified the microarray result by qRT-PCR,mi RNAs with high abundance and changed more than 2 folds were selected for further study,also,potential lncRNA-ATB binding sites were identified.In EMT cell model,we found over-expressed miR-200c by miR-200c mimic played similar role to lncRNA-ATB knockdown,they both reversed TGF-?1 induced EMT process,indicated that miR-200c also involves in lung epithelium EMT.To verify the directly binding between lncRNA-ATB and miR-200c,we predicted the binding site through the bioinformatics software.Then,dual luciferase reporter gene assay and RNA pull-down were performed,the results were supportive.These results proved lncRNA-ATB could sponge miR-200c during EMT.3.miR-200c attenuates silica-induced pulmonary fibrosisTo further verify the involvement of miR-200c during silicosis,we established silica-induced pulmonary fibrosis mice model.Lung tissues of silica-treated for 0,7,14,or 28 days were chosen to measure expression levels of miR-200c and other markers.The immunohistochemistry assays showed Collogen I levels increased with longer treatment,protein levels of E-cadherin decreased by Western Blot,while Vimentin,ZEB1,Fibronectin,and?-SMA levels increased significantly.Meanwhile,expression levels of miR-200c reduced gradually.Then,we over-expressed miR-200c levels by using mi R-200c agomir in mice.The results suggested that compared with silica combined with miR-NC agomir-treated mice,miR-200c agomir successfully elevated miR-200c level in mice lung.We used HE staining by using lung tissue pathology section.The experiment together with fibrosis score proved miR-200c agomir alleviate silica-inducd pulmonary fibrosis in mice,which was supported by related EMT and fibrosis markers.4.LncRNA-ATB sponged miR-200c targets ZEB1 to regulate EMT process of lung epitheliumFormer studies demonstrated that mi R-200c involve in EMT process by targeting ZEB1 mRNA 3'UTR region,which is an EMT inducing factor.So we treated TGF-?1 stimulated with miR-200c mimic or miR-NC mimic.Overexpression of miR-200c reduced both mRNA and protein levels of ZEB1.Dual luciferase reporter gene assays supported miR-200c targeting ZEB1 directly.After proved that lncRNA-ATB binding with mi R-200c and miR-200c targeting ZEB1 to play its role,we performed combined inhibition and function rescue experiments to further verify the signaling pathway.Firstly,we treated TGF-?1-stimulated Beas-2B cells with lncRNA-ATB siRNA and miR-200c mimic separately or together.We found after treated with both siRNA and mimic,the lncRNA-ATB levels decreased,while miR-200c levels elevated,the mRNA and protein levels of ZEB1 were inhibited significantly,and EMT marked showed most significant change compared with other groups.Indicating that down-regulated lncRNA-ATB and over-expressed miR-200c have combined inhibition effect during EMT process.Next,we used lncRNA-ATB siRNA with miR-200c inhibitor separately or together in TGF-?1 treated cells.QRT-PCR suggested miR-200c inhibitor could reverse lncRNA-ATB siRNA induced increase of mi R-200c,and also recovered levels of the target gene ZEB1 and EMT markers.Similarly,we transfected lncRNA-ATB plasmid with miR-200c mimic separately or together in normal cells.Over-expressed lncRNA-ATB promoted EMT,which could be inhibited by miR-200c mimic.Together,these results indicated that miR-200c could function as downstream of lncRNA-ATB,and reverse the lncRNA-ATB promoted EMT process.We proposed that lncRNA-ATB could regulate lung epithelium EMT by sponging miR-200c to recover expression of ZEB1.5.Silica-stimulated macrophages promote EMT process of lung epithelium by secreting TGF-?1In the process of silica-induced pulmonary fibrosis,macrophages could secrete a mount of TGF-?1 to play its pro-fibrogenic effect in the cellular environment.To figure out the upstream resource of elevated lncRNA-ATB in EMT process of lung epithelium,we used 0-200?g/m L suspended SiO₂ to stimulate PMA induced macrophages.The ELISA and Western Blot showed the synthesis and secretion levels of TGF-?1 reached to the top after treated with 150?g/mL SiO₂.We cultured Beas-2B cells by medium from SiO₂-treated macrophage.The result showed lncRNA-ATB increased while miR-200c decreased,and the Beas-2B cells underwent EMT process.To confirm the pro-EMT effect was performed by TGF-?1,we also added the TGF-?1 inhibitor SB 431542 into the medium.The inhibitor completely counteracted the effect of medium from SiO₂-treated macrophage,and levels of lncRNA-ATB and miR-200c returned to normal.Considering M2 macrophage is the main type of macrophages which secrete TGF-?1,we performed fluorescence activated cell sorting analysis by using M2 macrophage specific antigen CD206 to sort SiO₂-treated macrophages.Then we collected the sorted M2 macrophages to co-culture with Beas-2B cells.The co-culture significantly promoted EMT process of the lung epithelial cells as well as lncRNA-ATB.This part of study suggested SiO₂stimulate macrophages to polarized into M2 macrophages,provoked lung epithelial EMT by lncRNA-ATB.ConclusionsOur study proposed that during the process of silica-induced pulmonary fibrosis,SiO₂-stimulated macrophages secreted plenty of TGF-?1,which increased the level of lncRNA-ATB in lung epithelial cells.The elevated lncRNA-ATB promoted EMT process of pulmonary epithelium during silicosis by sponging miR-200c and retrieving miR-200c target gene ZEB1.Our subject would be helpful to complete the lncRNA-ATB related molecular mechanism and regulatory network in silica-induced pulmonary fibrosis.