LncRNA CHRF Sponges MiR-489 To Regulate MyD88 And Smad3 In Silica-induced Pulmonary Fibrosis

Silicosis is a chronic fibrotic lung disease caused by the long-term inhalation and deposition of crystalline silicon dioxide or silica in the lung.Silicosis is one of the most severe occupational diseases in China.Mounting evidence indicates that silica particles activate macrophages,causing them to release copious inflammatory and fibrotic cytokines,which in turn results in inflammatory cell infiltration,fibroblast proliferation,collagen,laminin and fibronectin secretion and extracellular matrix deposition in lung tissues.Meanwhile,injured alveolar epithelial cells could differentiate into myofibroblast through epithelial-mesenchymal transition(EMT),and ultimately pulmonary fibrosis.Though the pathogenic factor of silicosis is clear,the complex biological and molecular mechanisms underlying silicosis have not yet been fully elucidated,which hinder the development of novel therapeutic targets for silicosis.Therefore,new insights into the molecular mechanisms underlying the development of silicosis are basis for resolving therapeutic problems of silicosis.Numerous studies about mammalian transcriptome have estabished that tens of thousands of sequences are transcribed into transcripts(about 87.3%).However,less than 3%of the transcripts encode proteins.A large part of the transcripts do not encode protein,which exert biological functions at the RNA level and play precise roles in regulating gene expression.These are defined as non-coding RNA(ncRNA),including microRNA,lncRNA,siRNA,piRNA,snoRNA and so on.MicroRNA(miRNA)play a key role in a broad range of biological processes by pairing to complementary sequences in mRNA 3' untranslated regions(UTRs)of protein-coding genes and inhibiting the translation of or promoting the degradation of target mRNA.Recently,evidence indicates that some miRNAs are involved in pulmonary fibrosis by regulating multiple targets and signaling pathways;however,the current understanding of their roles in silicosis remains limited.Our previous miRNA microarray study has shown that miR-489 expression is decreased in lung tissues harvested on different days after the silica injection.In this study,we further clarified the roles and molecular mechanisms of miR-489 in silica-induced pulmonary fibrosis.Long non-coding RNAs(lncRNAs)are non-protein coding transcripts longer than 200 nucleotides,which can regulate gene expression at the epigenetic,transcriptional and post-transcriptional levels.Studies indicate that lncRNAs are associated with the progression of multiply diseases,including cardiovascular disease,nervous system disease and organ fibrotic disease.To date,only a small portion of lncRNAs have been investigated regarding their functions in pulmonary fibrosis,most of them are in bleomycin induced lung fibrosis.Therefore,whether lncRNAs play important roles in silica-induced pulmonary fibrosis are needed to be further investigated.One previous study has shown that IncRNA CHRF sponge miR-489 to regulate cardiac hypertrophy processes.In this study,we further investigated whether lncRNA CHRF has similar effects on miR-489 and regulats silica-induced pulmonary fibrosis.ObjectiveThe aim of our study was to confirm the potential roles of miR-489 in silica-induced pulmonary fibrosis by upregulating miR-489 levels in vivo,and to clarify the complex molecular mechanisms of lncRNA CHRF,miR-489,inflammatory pathway molecule MyD88 and TGF-? pathway molecule Smad3 in silica-induced mouse macrophages and TGF-?1-induced fibroblasts models,and ultimately reveal that IncRNA CHRF sponges miR-489 to regulate inflammatory and fibrotic signaling pathways,thus modulating silica-induced pulmonary fibrosis.These may provide scientific basis for novel therapeutic targets for silicosis.MethodsWe re-established a mouse model of silica-induced pulmonary fibrosis.qRT-PCR analysis were used to examine the expression levels of miR-489 in mouse fibrotic lung tissues on days 7,14,and 28 after silica injection.The miR-489 mimic(agomir)were injected via the tail vein in order to establish up-regulated miR-489 mouse model.Histological examinations of the lungs were employed.Western blot analyses were used to observe the protein expression of inflammatory and fibrotic markers in mouse lung tissues.The luciferase reporter assays were used to identify miR-489 potential targets MyD88 and Smad3.The macrophages(RAW264.7,THP-1)treated with silica and fibroblasts(NIH/3T3,MRC-5)treated with TGF-?1 were used to observe miR-489,MyD88,Smad3,p-Smad3,inflammatory and fibrotic marker levels.miR-489 mimic or MyD88/Smad3 siRNA were transfected respectively,miR-489 mimic and MyD88/Smad3 plasmids were co-transfected into macrophages treated with silica and fibroblasts treated with TGF-?1.Western blot analyses were used to observe the regulation of miR-489 on MyD88 and Smad3 as well as inflammatory and fibrotic marker levels.The fluorescence in situ hybridization(FISH)analysis was used to determine the cellular localization of IncRNA CHRF in macrophages and fibroblasts.A luciferase reporter assay and RNA pull down assay were used to determine whether IncRNA CHRF combines to miR-489 directly.lncRNA CHRF plasmids and siRNA were transfected respectively,lncRNA CHRF plasmids and MyD88/Smad3 siRNA were co-transfected,miR-489 mimic and IncRNA CHRF plasmids/siRNA were co-transfected into macrophages treated with silica and fibroblasts treated with TGF-?1.Western blot analyses were used to observe the regulation of IncRNA CHRF and miR-489 on MyD88 and Smad3 in silica-induced pulmonary fibrosis.Results1.miR-489 attenuates silica-induced pulmonary fibrosis in vivoBased on our previous microarray data,we chosed miR-489 for the further study.First,we re-established a mouse model of silica-induced pulmonary fibrosis and examined the expression levels of miR-489 in fibrotic lung tissues.We confirmed that the miR-489 levels on days 7,14,and 28 after silica injection were decreased compared with those in the saline group.Here,we hypothesized that the overexpression of miR-489 could attenuate silica-induced pulmonary fibrosis.We injected the miR-489 mimic(agomir)to restore the levels of miR-489 in vivo.Histological examination via hematoxylin and eosin(H&E)staining and fibrosis score on days 28 after silica injection showed that both the severity and distribution of lung lesions were ameliorated after miR-489 agomir treatment.Furthermore,the silica-induced elevations in the protein expression of a-SMA and Vimentin were significantly reduced in the presence of miR-489 agomir.Additionally,the protein expression of E-cadherin was restored in the miR-489 agomir treatment group.Together,these results confirmed that the overexpression of miR-489 attenuates silica-induced pulmonary fibrosis in vivo.2.miR-489 suppresses the initial inflammation of silica-induced pulmonary fibrosis by targeting MyD88Having demonstrated that miR-489 attenuates silica-induced pulmonary fibrosis in vivo,we sought to identify the potential targets of miR-489 by using bioinformatics databases method.One predicted target of miR-489 is MyD88,which is a key downstream adaptor for most Toll-like receptors.A luciferase reporter assay showed that the relative luciferase activity of MyD88-wt in macrophages was significantly reduced,whereas the MyD88-mut was unaffected.These data suggested that miR-489 might target MyD88 and thereby inhibit the initial inflammatory signaling events in silica-induced pulmonary fibrosis.IL-10 is one of the downstream effectors of the MyD8 8-dependent Toll-like receptors signaling pathway,and fibrogenic factor TGF-?1 are primarily derived from activated and impaired macrophages.We thus hypothesized that the decreased miR-489 might affect the IL-1? level by increasing the expression of MyD88 and then triggering an inflammatory response,thus further influencing TGF-?1 release directly or indirectly.We observed that MyD88;IL-1? and TGF-?1 protein levels in fibrotic lung tissues exhibited an increasing trend after silica treatment in mouse models.In contrast,the overexpression of miR-489 in a mouse model appeared to decrease MyD88,IL-1? and TGF-?1 levels.We treated macrophages with silica and observed that miR-489 levels rapidly decreased.As expected,miR-489 overexpression decreased the protein expression levels of MyD88,IL-1? and TGF-?1 in silica-induced macrophages.The treatment with siRNA against MyD88 in silica-induced macrophages resulted in decreased IL-1? and TGF-?1 protein expression.Moreover,MyD88 plasmids transfected into silica-induced macrophages could reverse the anti-inflammatory effects of miR-489.These data indicated that miR-489 directly targets MyD8 8 and thereby inhibits initial inflammation of silica-induced pulmonary fibrosis.3.miR-489 suppresses silica-induced fibrosis process by targeting Smad3Using bioinformatics databases method,we focused on another predicted target of miR-489,Smad3,a key molecule in the TGF-? pathway.A luciferase reporter assay showed that the relative luciferase activity of Smad3-wt in fibroblasts was significantly reduced,whereas the Smad3-mut was unaffected.These data suggested that miR-489 might suppress silica-induced fibrosis process by targeting Smad3.Then,we found that total Smad3 and p-Smad3 expression both increased in fibrotic lung tissues after silica injection.In contrast,down-regulated levels of total Smad3 and p-Smad3 were observed in a miR-489 overexpression mouse model.Furthermore,miR-489 levels were significantly decreased in fibroblasts treated with TGF-?1,and the protein levels of total Smad3,p-Smad3,?-SMA and Vimentin were increased compared with those in the control group,and the overexpression of miR-489 via mimic transfection led to repressed levels of total Smad3 and p-Smad3 and reduced the a-SMA and Vimentin levels in TGF-?1-induced fibroblasts.Next,the siRNA against Smad3(siSmad3)was used in TGF-?1-induced fibroblasts.We found that the protein expression levels of a-SMA and Vimentin were inhibited by siSmad3 transfection in fibroblasts.Additionally,Smad3 plasmids transfected into TGF-?1-induced fibroblasts could reverse the anti-fibrotic effects of miR-489.Overall,these data indicated that miR-489 suppresses the fibrotic process by targeting Smad3.All these findings provided further evidence of miR-489 as an important negative regulater that targets MyD88 and Smad3,and then blocks the initial inflammation and subsequent silica-induced fibrotic process.4.lncRNA CHRF negatively regulates miR-489 expression levelsNumerous lncRNAs have been reported to regulate the molecular functions of miRNAs.One study has shown that IncRNA CHRF represses miR-489 levels in cardiac hypertrophy.To investigate whether lncRNA CHRF plays a similar role in silica-induced pulmonary fibrosis,we performed qRT-PCR analysis to determine the lncRNA CHRF levels and found that lncRNA CHRF was significantly increased in mouse fibrotic lung tissues,the silica-induced macrophages and TGF-?1-induced fibroblasts.These results indicated that lncRNA CHRF may be crucial in silica-induced pulmonary fibrosis.Subsequently,we performed the fluorescence in situ hybridization(FISH)analysis and observed that lncRNA CHRF was located both in cytoplasm and nucleus of macrophages and fibroblasts,A luciferase reporter assay and RNA pull down assay showed that lncRNA CHRF could combine to miR-489 directly.To explore the possibility that IncRNA CHRF regulates miR-489 expression,macrophages and fibroblasts were transfected with miR-489 mimic and then engineered to stably over-express IncRNA CHRF via CHRF plasmids;knockdown of IncRNA CHRF was achieved via siCHRF.As expected,the overexpression of IncRNA CHRF reversed the elevation of miR-489 levels compared with the levels in the miR-489 mimic group in silica-induced macrophages and TGF-?1-induced fibroblasts.In contrast,knockdown of IncRNA CHRF promoted the elevation of miR-489 levels in two cell lines.Thus,these results suggest that lncRNA CHRF negatively regulates miR-489 expression.5.lncRNA CHRF promotes silica-induced pulmonary fibrosis through sponging miR-489Based on the regulation of IncRNA CHRF on miR-489 expression,we next determined whether IncRNA CHRF is associated with the progression of silica-induced pulmonary fibrosis.We found that the overexpression of lncRNA CHRF induced significant increases in MyD88,Smad3 and p-Smad3 protein levels as well as inflammatory and fibrotic marker levels in macrophages and fibroblasts.Knockdown of MyD88 and Smad3 inhibited those roles of IncRNA CHRF,suggesting that MyD88 and Smad3 are downstream targets of IncRNA CHRF.Furthermore,the knockdown of lncRNA CHRF inhibited the increases in MyD88,Smad3 and p-Smad3 levels as well as inflammatory and fibrotic marker levels in macrophages and fibroblasts.Together,our results indicated that IncRNA CHRF regulates MyD88 and Smad3,and consequently promotes the silica-induced initial inflammation and subsequent fibrotic process.To confirm that lncRNA CHRF regulates MyD88 and Smad3 by sponging miR-489 in the pulmonary fibrosis network,the co-transfection methods were used in following experiments.When co-transfecting miR-489 mimic and lncRNA CHRF plasmid,we observed that the overexpression of IncRNA CHRF counteracted the repression of miR-489 on its target genes(MyD88,Smad3)and increased IL-1?,TGF-?1,?-SMA and Vimentin expression.Moreover,after co-transfecting miR-489 mimic and siCHRF,we found that the knockdown of IncRNA CHRF enhanced the roles of miR-489 in inflammatory and fibrotic pathways.These results suggested that IncRNA CHRF,by sponging miR-489,regulates its targets MyD88 and Smad3 and participates in silica-induced pulmonary fibrosis.ConclusionsIn this study,our findings indicated that miR-489 attenuates silica-induced pulmonary fibrosis in vivo and in vitro.The molecular study demonstrated that miR-489 attenuates silica-induced pulmonary fibrosis primarily by repressing its target genes MyD88,an important molecule of inflammatory pathway,and Smad3,a key molecule in the TGF-? pathway.Further studies have shown that IncRNA CHRF sponged miR-489 and reversed the inhibitory effect of miR-489 on MyD88 and Smad3,then triggering the inflammatory and fibrotic signaling pathways.Overall,our data indicated that IncRNA CHRF-miR-489-MyD88 and lncRNA CHRF-miR-489-Smad3 signaling axis exert key functions in silica-induced pulmonary fibrosis.