LncRNA-ATB Promotes EMT In Silica-induced Pulmonary Fibrosis Through MiR-29b-2-5p And MiR-34c-3p

Silicosis,an irreversible progressive disease characterized by pulmonary fibrosis,is a major type of pneumoconiosis due to inhalation of free silica dust.The pathological process of silicosis is complicated in which silica exposure activates lung macrophages,damages alveolar epithelial cells,promotes epithelial-mesenchymal transition and triggers fibroblast activation and proliferation.This process involves multiple stages including oxidative stress,immune response,inflammatory response and fibrosis formation,which leads to extracellular matrix（ECM）deposition and alveolar structure destruction.Currently,though some gratifying progress has been made in the molecular mechanism and intervention researches of silicosis,however,there are still no effective treatments.Epithelial-to-mesenchymal transitionis（EMT）is a dynamic and reversible biological process during which epithelial cells lose their cell polarity,down-regulate intercellular adhesion.It inhibits the expression of epithelial conniving along with increasing expression of certain mesenchymal proteins leading to a shift from epithelial morphology and physiology to the mesenchymal phenotype to gain migration properties.Studies have shown that EMT participates in various processes,such as cancer metastasis and fibrosis.It has been reported that EMT also played an important role in silica-induced pulmonary fibrosis and our previous research has confirmed this,but the exact molecular mechanism needs further exploration.Long non-coding RNA（lncRNA）is a class of functional RNAs that regulates a variety of biological functions through gene networks including epigenetic,transcriptional and post-transcriptional regulation.Dysregulated lncRNA expression has been observed in multiple pathological conditions,tissue fibrosis for instance,showing that it may play an important role in progression of disease.It is reported that lncRNA is involved in the development of EMT through binding to its corresponding miRNA to regulate tissue fibrosis.LncRNA-ATB,a long non-coding RNA activated by TGF-β,plays an important role in TGF-βsignaling pathway.Currently,researches have found that lncRNA-ATB participates in the progress of a variety of tumors.It regulates metastasis and invasion of cancer cells through adjusting EMT induced by TGF-β,but the role of lncRNA-ATB in silicosis has not been fully understood yet.We previously conducted miRNA array by knocking down the expression of lncRNA-ATB,and confirmed that lncRNA-ATB could regulate EMT process through competitively adsorption of miR-200c to influence the progress of silica-induced pulmonary fibrosis.Of our particular interest,based on the miRNA array analysis,we found that the expression of several miRNAs were significantly dysregulated besides miR-200c.In this study,we identified miR-29b-2-5p and miR-34c-3p as two new attractive targets of lncRNA-ATB.Consistent with the result of miRNAs array,the expression of miR-29b-2-5p and miR-34c-3p were significantly increased with the silence of lncRNA-ATB.Therefore,the study was to further investigate whether lncRNA-ATB regulated EMT process through sponging miR-29b-2-5p and miR-34c-3p in silica-induced lung fibrosis to provide theoretical basises as well as potential therapeutic targets for the prevention and treatment of silicosis.ObjectiveAccording to our previous study,we aimed to explore whether miR-29b-2-5p and miR-34c-3p were two new target miRNAs of lncRNA-ATB trying to figure out the regulatory relationship between lncRNA-ATB and miR-29b-2-5p or miR-34c-3p via in vitro experiemts.We also overexpressed miR-29b-2-5p and miR-34c-3p alone or together to investigate their inhibitory effect in vivo,so as to find new strategies for the treatment of silicosis.Methods1.LncRNA-ATB overexpression plasmid and siRNA were transfected in cells treated with TGF-β1 to detect the expression of miR-29b-2-5p and miR-34c-3p by q RT-PCR.Dual luciferase reporter assays、RIP and RNA pull-down experiments were used to verify the binding between lncRNA-ATB and the two miRNAs.2.A549 and Beas-2B cells were treated with TGF-β1 to establish EMT models.QRT-PCR was used to detect miR-34c-3p and miR-29b-2-5p expression.Western Blot was used to detect expression of fibrosis and EMT markers.miR-34c-3p and miR-29b-2-5p mimics were used to detect the expression of fibrosis and EMT markers.3.Lnc RNA-ATB si RNA and miR-29b-2-5p or miR-34c-3p inhibitor were co-transfected to further confirmed the regulatory relationship through detection of EMT and fibrosis markers.4.The targets of miR-34c-3p and miR-29b-2-5p were predicted using bioinformatics websites and were confirmed by double luciferase reporter gene experiment.Western blot was to observe the expression of MEKK2 and Notch2 in TGF-β1 induced EMT models or in miR-34c-3p and miR-29b-2-5p overexpressed cells.5.Lung fibrosis mouse model was constructed through intratracheal instillation with Si O₂suspension;the intervention mouse model was established by tail vein injection of miR-34c-3p or miR-29b-2-5p agomir;joint intervention mouse model was constructed using intratracheal instillation of miR-34c-3p and miR-29b-2-5p adenovirus alone or together for three weeks and then intratracheal instillation with Si O₂.HE staining was to assess the degree of fibrotic lesions;the combined inhibition of EMT and fibrosis markers was measured by western blot and hydroxyproline content;q RT-PCR was used to measure expression of miR-34c-3p and miR-29b-2-5p.Results1.LncRNA-ATB binds with miR-34c-3p and miR-29b-2-5pWe found that miR-29b-2-5p and miR-34c-3p might be two new target miRNAs of lncRNA-ATB based on our early miRNA array.After the transfection with lncRNA-ATB si RNA or plasmid,the expression levels of miR-29b-2-5p and miR-34c-3p increased or decreased accordingly in the TGF-β1 treated A549 cells.Further,we found the potential binding sites between lncRNA-ATB and the two miRNAs by bioinformatics analysis and verified the binding by the dual luciferase reporter gene assay,RIP and RNA pull-down experiments.2.Lnc RNA-ATB promotes EMT through regulation of miR-34c-3p and miR-29b-2-5p.A549 and Beas-2B cells were treated with TGF-β1 to establish EMT models.In the cell models,we observed that lncRNA-ATB was significantly elevated but miR-29b-2-5p and miR-34c-3p were down regulated.Then through intracellular transfection of miR-34c-3p or miR-29b-2-5p mimic,we found the increased level of E-cadherin along with decreased expression of Vimentin,Fibronectin andα-SMA in TGF-β1 treated A549 and Bease-2B cells,indicating that overexpression of miR-34c-3p and miR-29b-2-5p blocked the progression of EMT in lung epithelial cells.Moreover,progression of EMT was also inhibited when down-regulated the expression of lncRNA-ATB.Besides,we co-transfected lncRNA-ATB si RNA and miR-34c-3p or miR-29b-2-5p inhibitor,which showed that the suppressed EMT process was rescued to some extent,indicating that lncRNA-ATB could sponge miR-34c-3p and miR-29b-2-5p to regulate EMT in epithelial cells.3.MEKK2 and Notch2 are target genes of miR-29b-2-5p/miR-34c-3p.After predicting the target genes of miR-34c-3p and miR-29b-2-5p through bioinformatics softwares,the binding between miR-34c-3p and MEKK2 as well as Notch2 or between miR-29b-2-5p and MEKK2 were verified by double luciferase reporter gene experiments.In TGF-β1 indecued EMT models,the expression of MEKK2 and Notch2 were significantly increased compared with the control group,while they were obviously decreased with the transfection of miR-34c-3p and miR-29b-2-5p mimic.These results revealed that miR-34c-3p and miR-29b-2-5p regulated EMT through targeting MEKK2 and Notch2.4.miR-34c-3p and miR-29b-2-5p are down-regulated in silica induced mice pulmonary fibrosisHE staining of pulmonary pathological section showed the formation of diffuse pulmonary fibrosis and the degree of lung fibrosis got serious with silica treatment.Simultaneously the content of hydroxyproline increased after treatment with silica.And the expression of epithelial marker（E-cadherin）decreased,however,extracellular matrix（Fibronectin）and mesenchymal markers（α-SMA,Vimentin）levels increased in a time-dependent manner along with gradually decreased levels of miR-34c-3p and miR-29b-2-5p.5.miR-34c-3p and miR-29b-2-5p attenuate silica-induced pulmonary fibrosisWe observed the effect of miR-34c-3p and miR-29b-2-5p on mouse pulmonary fibrosis through intravenous injection of miR-34c-3p and miR-29b-2-5p agomir.The results showed that the overexpression of miR-34c-3p and miR-29b-2-5p reduced the protein expression of EMT and fibrosis markers,inhibited the content of hydroxyproline in mouse lungs and ameliorated the damaged alveolar structure which was consistent with the fibrosis score analysis.All results indicated that miR-34c-3p and miR-29b-2-5p could attenuate silica-induced mouse pulmonary fibrosis.6.miR-29b-2-5p and miR-34c-3p have a combined inhibitory effect on EMT and pulmonary fibrosisThe lung epithelial cells were transfected with miR-34c-3p mimic and miR-29b-2-5p mimic alone or together.The protein levels revealed that,compared with transfection alone,the co-transfection group showed a higher expression of E-cadherin and a lower expression of vimentin,fibronection andα-SMA,indicating that the two miRNAs might have a combined effect on the inhibition of EMT.Consistent with the cellular results,the adenovirus mouse models showed that overexpression of miR-34c-3p or miR-29b-2-5p inhibited silica-induced lung fibrosis seperately.Moreover,it showed more stronger inhibitory effect on fibrosis when over-expressed miR-34c-3p and miR-29b-2-5p simultaneously.These resluts suggested that miR-34c-3p and miR-29b-2-5p inhibited EMT in cooperation in lung epithelial cells and alleviated lung fibrosis in mice.ConclusionOur study demonstrated that miR-34c-3p and miR-29b-2-5p participated in the regulation of EMT process through MEKK2 and Notch2 signal pathway,which was regulated by lncRNA-ATB.In addition,miR-34c-3p and miR-29b-2-5p had a combined inhibitory effect on fibrosis in this process.