CDR1as Regulates EMT To Promote Silica-induced Pulmonary Fibrosis Via Sponging MiR-7

Pneumoconiosis is a systemic disease featured with diffuse fibrosis of lung tissue,mainly caused by occupational inhalation of productive dust.Silicosis is one of the typical forms of pneumoconiosis,characterized by excessive accumulation of fibroblasts and pathological remodeling of extracellular matrix?ECM?.Exposure of silica causes activation of various effector cells and secretion of cytokines and inflammatory mediators,which induce epithelial cell transformation?EMT?,myofibroblasts and fibroblasts proliferation and ECM over-deposition.As a result,normal alveolar structure will be destroyed,which lead to organ dysfunction or even death.There are complex biological and molecular mechanisms underlying silicosis,which remains not clear.Growing evidences suggest that EMT plays a crucial role in fibrotic diseases including silicosis.Epithelial-mesenchymal transition?EMT?refers to the biological process that epithelial cells transform into interstitial cells through specific procedures.According to previous findings,alveolar epithelial cells undergoing EMT are considered as potential sources of myofibroblasts,which provides convincing evidence for the crucial role of EMT in the development of pulmonary fibrosis.In injury or remodeling of tissue,the EMT process of alveolar epithelial cells will be activated.During the EMT process,epitheliums lose their epithelial characteristics like polarity,cell-cell adhesion and high expression of epithelial markers?E-cadherin,zonula occludens-1?,meanwhile acquire a migratory behavior and mesenchymal phenotype including high levels of?-smooth muscle actin??-SMA?,Collagen I and Vimentin.MicroRNAs?miRNAs?are small?18²5 nucleotides?,highly conserved non-coding RNA molecules.miRNAs act as the gene regulators by binding to the3-untranslated region?UTR?of their target mRNAs to inhibit their translation or mediate their degradation.These non-coding RNAs play pivotal roles in multiple cellular processes like cell proliferation,apoptosis and invasion.Recent studies demonstrated that dysregulation of miRNAs was linked to the susceptibility of diseases,play pivotal roles in lung diseases,including asthma,lung cancer and pulmonary fibrosis.Several miRNAs have been reported to regulate the process of lung fibrosis,and miRNAs silence or over-expression may become a potential strategy for the treatment of fibrotic diseases.Circular RNAs?circRNAs?are newly emerging endogenous noncoding RNAs,which have attracted much attention in recent years.Recent research has revealed that circRNAs are important regulators and modifiers of gene expression,and one of its major function is direct combination with miRNAs as“miRNA sponges”.However,the function of circRNAs in lung fibrosis is rarely reported,which highlights the need to identify aberrant circRNAs and to clarify their general mechanism in pulmonary fibrosis.CDR1as,also known as CiRS-7,is antisense to cerebellar denaturation related protein transcripts.It was reported that CDR1as could play a regulatory role in a variety of diseases by acting as miR-7 sponges.In lung fibrosis,the effect of CDR1as on miR-7 needs to be further explored.Objective:The pulmonary fibrosis model of mice and EMT cell model were established by using silica dust.At the same time,we intervened the expression of miR-7 in vivo and vitro,in order to explore the potential regulatory effect of miR-7on EMT process in silica-induced pulmonary fibrosis.In addition,the expression of CDR1as was intervened at cellular level to explore its effect on miR-7 and EMT regulation,so as to partly reveal the pathogenesis of silicosis and provide a new insight for new prevention and control strategies for silicosis.Methods:?1?Pulmonary fibrosis was induced by intratracheal instillation of SiO₂.The mice were divided into four groups,including silica dust for 0 days,7days,14 days and 28 days.The lung pathological changes of mouse lungs was evaluated by pathological section with HE staining.The changes of fibrosis and EMT markers were detected by Western blot.?2?EMT cell models of HBE and A549 cells were established by SiO₂ treatment and morphological changes of them were observed under microscope.Western blot was performed to detect the protein level of fibrosis and EMT markers.?3?qRT-PCR was performed to determine the expression level of miR-7 in both mice and cell models.?4?The mice and epithelial cells were co-treated with silica and miR-7 agomir or miR-7 mimic.And then miR-7expression was detected via qRT-PCR,the changes of fibrosis and EMT related proteins were determined via western blot.?5?TGFBR2 was predicted to be the target gene of mi R-7 by using bioinformatics softwares,which was verified via double luciferase reporter gene assay,and Western blot wass performed to observe the effect of miR-7 over-expression on the level of TGFBR2 and downstream molecules p-Smad2/3.?6?It was predicted that CDR1as could sponge miR-7,which has been confirmed in other studies.Then CDR1as was knocked down alone by using siRNA and miR-7 changes were detected via qRT-PCR.Western blot was performed to observe the expression level of TGFBR2,p-Smad2/3 and EMT related proteins.Then the regulatory relationship between CDR1as and miR-7 was verified through co-administration of CDR1as and miR-7 inhibitor.?7?Lung fibroblasts were treated with miR-7 mimic and TGF-?1,then fibrosis markers and the proliferation of cells were detected.Results:1.miR-7 is down-regulated in lung tissues during silica-induced pulmonary fibrosisAfter treatment with silica for 28 days,formation of diffuse pulmonary fibrosis and fibrotic nodules appeared.The protein levels of epithelial marker E-cadherin decreased,while fibrotic markers Collagen I,?-SMA and CTGF were significantly increased in mice lung tissues.Next the level of miR-7 was determined and miR-7was confirmed to be gradually down-regulated after silica exposure.These results suggested that miR-7 was decreased in silica-induced pulmonary fibrosis,and EMT process was activated.2.miR-7 attenuates silica-induced pulmonary fibrosis in vivoThe histological examination revealed that mice in silica group tended to develop more extensive and severe lung lesions compared with those in silica+miR-7 agomir group,which was consistent with the results of fibrosis score.In addition,the up-regulated protein levels of Collagen I,?-SMA and CTGF were significantly reversed by over-expression of miR-7.Also,increased level of miR-7enhanced the expression of E-cadherin compared with silica+miR-NC group.These results demonstrated that over-expression of miR-7 may play an anti-fibrotic effect through inhibiting EMT process in silica-induced pulmonary fibrosis.3.miR-7 inhibits EMT process induced by silicaEMT models were successfully set up in HBE and A549 cells by treatment of silica.In the EMT process of pulmonary epithelial cells,the synthesis of miR-7 was reduced in a time and dose manner.After restoring miR-7 by using miR-7 mimic in silica-induced EMT cells?Fig.3D?,the changes of EMT related proteins??-SMA,CTGF and E-cadherin?were repressed.These data suggested that miR-7 could impede the process of EMT induced by silica.4.TGFBR2 is a downstream target of miR-7 during silica-induced EMT processThe potential target genes of miR-7 were identified by using publicly available bioinformatics databases.Then it was found that TGFBR2,an important receptor of TGF-?1,contained potential binding sites of miR-7,which was verified via a dual luciferase reporter gene assay.The protein levels of TGFBR2 were significantly increased in the silica-treated lung tissues of mice and EMT cell models,which was contrary to the expression trend of miR-7.But miR-7 over-expression repressed the protein level of TGFBR2 and p-Smad2/3 up-regulated by silica both in vivo and vitro.These data indicated that TGFBR2 was a direct target of miR-7 during EMT in silica-induced pulmonary fibrosis.5.CDR1as serves as a EMT regulator via suppression of miR-7 in silica-induced pulmonary fibrosisThrough bioinformatics software prediction,it was found that a circRNA CDR1as has multiple complementary sequences that match the seed sequence of miR-7,which have been verified in papers.In contrast to the decrease of miR-7,the increase of CDR1as was observed after treatment of silica,indicating that it may be involved in EMT process in pulmonary epithelial cells.Our results showed that CDR1as was enriched in the cytoplasm via fluorescence in situ hybridization?FISH?assay and isolation of cytoplasmic and nuclear RNA,which is an important character of ceRNA.Then we inhibited silica-induced CDR1as up-regulation via CDR1as siRNA and observed that CDR1as siRNA significantly reversed EMT,which was induced by silica in HBE and A549 cells.Additionally,CDR1as knockdown decreased the elevated levels of TGFBR2 and p-Smad2/3 induced by silica in both cells.These results indicated that CDR1as inhibit the suppressive effect of miR-7 on EMT through regulating TGFBR2.To further confirm the regulatory role of CDR1as,co-transfection experiments were performed in the following study.The suppressive function of up-regulated miR-7 on EMT and TGF-?1/Smad signal pathway was counteracted in the presence of miR-7 inhibitor.All the results suggest that CDR1as promoted EMT process through modulating miR-7 and its target TGFBR2 in silica-induced pulmonary fibrosis.6.miR-7 represses activation of lung fibroblasts induced by TGF-?1TGF-?1 was found to down-regulate the expression of miR-7 at the dose of5ng/ml and 10ng/ml in MRC-5 and NIH/3T3 cells.After restored miR-7 in the cells,elevated levels of Collagen I,?-SMA and CTGF induced by TGF-?1 were significantly decreased compared with mimic NC group.Similar changes of Collagen I,?-SMA and CTGF were observed in vivo.In addition,miR-7 had an inhibitory effect on TGF-?1-induced proliferation of lung fibroblasts.These data showed that mi R-7 may repress activation of lung fibroblasts induced by TGF-?1.Conclusion:We confirmed that mi R-7 could inhibit EMT and activation of lung fibroblasts to play its anti-fibrotic role.Moreover,CDR1as could act as a pro-fibrotic molecule and repress the inhibitory effect of miR-7 on EMT through sponging miR-7.Our study revealed that the interaction between miR-7 and CDR1as may play an important role to regulate EMT process in lung fibrotic diseases.