The Molecular Mechanism Of FOXM1/lncRNA-PVT1/miR-497-5p Axis In Silica-induced Pulmonary Fibrosis

Introduction Silicosis is a pulmonary disease caused by long-term inhalation of silica dust during occupational activities,which is mainly characterized by irreversible fibrosis of the lung tissues.It is one of the common types of pneumoconiosis and is the most widespread and harmful occupational disease in China.It is expected that in the next 20 years or more,pneumoconiosis(especially silicosis)will still bring a heavy economic burden to the society and individuals,and seriously affect the life quality of patients.Accordingly,silicosis is a critical public health problem that cannot be ignored in China.The pathogenesis of silicosis is complex and not yet fully understood.Current research suggests that the progress of silicosis involves different types of cells,multiple stages,and a very large number of cytokines.However,there is still no specific biomarkers or effective treatment for this disease currently.Therefore,an in-depth pathogenesis study of silicosis can provide a solid theoretical basis for its early diagnosis,intervention and treatment.The progression of silicosis mainly includes the early inflammatory stage and the late fibrotic stage.Myofibroblasts are the key players in the fibrotic stage.Activated fibroblasts are one of the main sources of myofibroblasts,and this activation process is an important event in pulmonary fibrosis,which is characterized by the abnormal deposition of the extracellular matrix,and can be regulated by transforming growth factor-β(TGF-β).Our study focused on the important event of lung fibroblast activation induced by TGF-β to deeply explore the potential regulatory molecules(such as non-coding RNA(nc RNA))and the possible regulatory mechanisms in the process of pulmonary fibrosis.Long noncoding RNA(lnc RNA)is a type of RNA with transcripts longer than 200 nucleotides.It participates in a variety of cell biological processes and is closely related to the development and prognosis of diseases.The molecular function of lnc RNA is tightly associated with its localization in cells.The cytoplasm-localized lnc RNA can interact with micro RNA(mi RNA)to influence the expression of the target m RNAs.Although some lnc RNAs have been reported to be linked with the progression of fibrosis,it is still unclear whether there are any lnc RNAs involved in the regulation of TGF-β-induced lung fibroblast activation during silica-induced lung fibrosis.To explore the key non-coding RNAs involved in the activation of lung fibroblasts,we performed lnc RNA sequencing in TGF-β1 treated and untreated human embryonic lung fibroblasts.A total of 28 lnc RNAs were found,of which the expression of 24 lnc RNAs increased after TGF-β1 treatment and 4 lnc RNAs decreased.Then these candidate lnc RNAs were further verified in the cell model,and the expression levels of 8 lnc RNAs were found to be consistent with the sequencing results,including lnc RNA-PVT1,NEAT1,DNM3 OS,etc.Among these lnc RNAs,lnc RNA-PVT1 showed great regulatory potential in diseases,but its research in pulmonary fibrosis is still missing,so we chose it for further study.Lnc RNA-PVT1(plasmacytoma variant translocation 1,plasmacytoma spliceosome 1)is the long non-coding RNA with 1957 nt.Existing research shows that lnc RNA-PVT1 plays a huge regulatory role by driving various biological effects,and has the potential to be a diagnostic,prognostic marker and therapeutic target in tumors.At present,there have been few studies on lnc RNA-PVT1 in fibrotic diseases,which have revealed that lnc RNA-PVT1 can participate in the process of organ fibrosis.However,whether lnc RNA-PVT1 can regulate silica-induced pulmonary fibrosis is still unknown.Further,the intracellular localization study of lnc RNA-PVT1 showed that it was abundantly expressed in the cytoplasm,suggesting that lnc RNA-PVT1 could competitively absorb mi RNAs to function.Combining bioinformatics analysis,literature search,and the results of the mi RNA array of the silica-induced pulmonary fibrosis in mice,we found that mi R-497-5p may interact with lnc RNA-PVT1,and further research was taken to study the potential function and specific molecular mechanism between them.It is reported that lnc RNA could be regulated by some transcription factors,for example,lnc RNA-PVT1 is positively regulated by FOXM1(Forkhead box M1)in gastric cancer cells.FOXM1 is a member of the forkhead box transcription factor family,which is closely related to the cell cycle and can drive the activation of lung fibroblasts.Our results also showed that the expression of FOXM1 was increased in the cell and mouse models,and knock down the level of FOXM1 affected the expression of lnc RNA-PVT1 and mi R-497-5p.Therefore,our study was to explore the profibrotic role of lnc RNA-PVT1 in regulating fibroblast activation during silica-induced pulmonary fibrosis,to reveal the underlying regulatory mechanisms among FOXM1,lnc RNA-PVT1,mi R-497-5p,so as to provide new clues for the prevention and treatment of silica-induced pulmonary fibrosis.Objective Through lnc RNA sequencing and cell function research,we confirmed the positive effect of lnc RNA-PVT1 on the activation of lung fibroblasts.Further,via the cellular experiments in vitro,we tried to explore the upstream transcription factors and the downstream mi RNAs of lnc RNA-PVT1 to reveal the molecular mechanism of lnc RNA-PVT1 in regulating the activation of lung fibroblasts;via the mouse models in vivo,we attempted to observe the intervention effect of the mi RNA sponged by lnc RNA-PVT1,so as to clarify the role of lnc RNA-PVT1 in silica dust-induced pulmonary fibrosis,which may lay the foundation for the prevention and treatment of silicosis.Methods(1)The cell model of lung fibroblast activation was constructed by treating MRC-5 cells with TGF-β1 in vitro.The mouse model of silica-induced pulmonary fibrosis was established via intratracheally instilling with silica particle suspension in vivo.The mouse model of mi R-497-5p agomir intervention was further established through the tail vein injection of mi R-497-5p.(2)High-throughput lnc RNA sequencing was used to search for abnormally expressed lnc RNAs in activated MRC-5 cells.(3)The location of lnc RNA-PVT1 in MRC-5 cells was investigated by isolation of cytoplasmic and nuclear RNA assay and fluorescence in situ hybridization experiments.(4)The dual-luciferase reporter gene assays and RNA pull-down experiment were used to verify the binding among lnc RNA-PVT1,mi R-497-5p,target m RNAs,and FOXM1.(5)The severity and distribution of mouse models were evaluated by HE staining and hydroxyproline content detection.(6)We observed the cell proliferation ability by the CCK8 assay;the cell migration ability by the wound-healing assay;the cell cycle by the flow cytometry experiment;the protein expression levels by Western Blot and the immunostaining experiment;the RNA expression levels by q RT-PCR.Results 1.Lnc RNA-PVT1 is involved in TGF-β1-induced lung fibroblast activation After establishing the activation cell model of lung fibroblasts via treating MRC-5 cells with TGF-β1,high-throughput lnc RNA sequencing was used to screen for differentially expressed lnc RNAs.Based on the principle that the differential expression was more than 2 times or between 1.5-2 times and the expression value(Count)> 50,we found 28 lnc RNAs with differential expression(P < 0.05).Next,the candidate lnc RNAs were further verified by q RT-PCR,and the results showed that the expression levels of the 8 lnc RNAs were consistent with the sequencing results.Combined with the literature review,we found that lnc RNA-PVT1 has great regulatory potential in disease,but its research in pulmonary fibrosis was still missing.Therefore,we selected lnc RNA-PVT1 for further study.Then lnc RNA-PVT1 si RNA was used to knock down the expression of lnc RNA-PVT1 in TGF-β1 treated MRC-5 cells,which showed a decreased expression of fibrotic proteins and a weakened cell migration capacity.These results demonstrated that lnc RNA-PVT1 could be involved in TGF-β1-induced lung fibroblast activation.2.The cytoplasm enriched lnc RNA-PVT1 competitively binds mi R-497-5p to function To explore the specific mechanism of lnc RNA-PVT1 involved in the activation of lung fibroblasts,the isolation of cytoplasmic and nuclear RNA assay and fluorescence in situ hybridization experiments were used to observe its localization in MRC-5 cells.The results showed that lnc RNA-PVT1 was abundantly expressed in the cytoplasm,suggesting a ce RNA function for lnc RNA-PVT1.Then the bioinformatics website was used to predict the mi RNA,and the dual-luciferase reporter gene assay and RNA pull-down experiment were used to confirm the binding between lnc RNA-PVT1 and mi R-497-5p.3.mi R-497-5p inhibits lung fibroblast activation in vitro To observe the regulatory effect of mi R-497-5p on lung fibroblasts,its expression level in the activated MRC-5 cells was first detected.q RT-PCR showed an obvious reduction of mi R-497-5p in TGF-β1 treated MRC-5 cells,thus we overexpressed its level by the mi R-497-5p mimic.TGF-β1-induced the forced protein expression of fibrotic markers was inhibited in the treatment with mi R-497-5p mimic.Consistently,immunofluorescence and wound healing experiments further demonstrated that mi R-497-5p upregulation could obstruct TGF-β1-induced MRC-5 activation.Similar results were also observed in mouse lung embryonic fibroblasts(NIH-3T3 cells).These data indicated that mi R-497-5p can inhibit lung fibroblast activation in vitro.4.mi R-497-5p attenuates silica-induced pulmonary fibrosis in vivo To investigate the function of mi R-497-5p in vivo,we first established a mouse model of silica-induced pulmonary fibrosis via intratracheally instilling with silica particle suspension.The expression level of mi R-497-5p was found to reduce significantly.Then,we over-expressed mi R-497-5p level in mice to construct an intervention model through tail vein injection of mi R-497-5p agomir.The results showed that mi R-497-5p agomir successfully increased the level of mi R-497-5p in the lung tissues of mice and significantly reduced the fibrotic score,hydroxyproline content and the protein expression of fibrosis markers in the mouse lung tissues,indicating that mi R-497-5p can reduce silica-induced pulmonary fibrosis in vivo.5.mi R-497-5p suppresses the activation of lung fibroblasts via targeting Smad3 and Bcl2 To better understand the role of mi R-497-5p in silica-induced pulmonary fibrosis,we searched for the potential targets by bioinformatics website prediction and dual-luciferase reporter gene experiments.It showed that Smad3 and Bcl2 were two target genes of mi R-497-5p.The RNA and protein expression levels of Smad3 and Bcl2 were suppressed with mi R-497-5p overexpression both in vitro and in vivo.We further transfected MRC-5 cells with si RNAs against Smad3 and Bcl2 respectively and found an inhibitory effect on the protein expression levels of fibrotic markers induced by TGF-β1.Moreover,we treated TGF-β1-stimulated MRC-5 cells with mi R-497-5p mimic and target gene plasmids,separately or together.Overexpression of target genes reversed the protein expression levels of fibrotic markers restrained by the mi R-497-5p mimic.These results revealed that mi R-497-5p could suppress TGF-β1-induced pulmonary fibroblast activation via targeting Bcl2/Smad3.6.Lnc RNA-PVT1 mediates Smad3 and Bcl2 through mi R-497-5p To figure out whether lnc RNA-PVT1/mi R-497-5p/Smad3 and Bcl2 axes are involved in TGF-β1-induced pulmonary fibroblast activation,combined inhibition and function rescue experiments were performed to further verify the signaling pathway.We first assessed the protein levels of Smad3 and Bcl2 in the presence of lnc RNA-PVT1 si RNA,which showed a repressed expression of the two target genes.Then,lnc RNA-PVT1 si RNA and mi R-497-5p mimic were transfected with TGF-β1 treated MRC-5 cells.The reduced expression of fibroblast activation markers suggested that both lnc RNA-PVT1 si RNA and mi R-497-5p mimic could rescue TGF-β1-induced the fibroblast activation,down-regulated lnc RNA-PVT1 cooperated with over-expressed mi R-497-5p in this process.Further,we investigate the resistance effect by using lnc RNA-PVT1 si RNA and mi R-497-5p inhibitor.Repressed lnc RNA-PVT1 decreased the levels of collagen I,fibronectin,α-SMA in TGF-β1 treated cells,while inhibition of mi R-497-5p reversed this effect.Additionally,the lnc RNA-PVT1 plasmid was constructed and found to induce fibroblast activation,however,combined incubated with mi R-497-5p mimic abolished the effect of lnc RNA-PVT1.These results demonstrated that lnc RNA-PVT1 could regulate Smad3 and Bcl2 through mi R-497-5p,leading to the activation of lung fibroblasts.7.FOXM1 acts as a profibrotic factor by elevating lnc RNA-PVT1 Studies have shown that lnc RNA-PVT1 is regulated by FOXM1,so we tested FOXM1 expression in vitro and in vivo models and found a significant elevation expression compared to controls.After knocking down the expression of FOXM1 in MRC-5 cells,it was found that the ability of cell proliferation and migration,as well as the fluorescence intensity of fibrosis activation markers were all weakened,indicating that FOXM1 was a positive regulator in the activation of lung fibroblasts.The dual-luciferase reporter gene assay confirmed that FOXM1 directly bound to the promoter region of lnc RNA-PVT1 to promote its transcription.Moreover,we found that the reduced expression of FOXM1 may decrease the level of lnc RNA-PVT1 and increase the level of mi R-497-5p,indicating that FOXM1 regulated the transcription of lnc RNA-PVT1,which in turn affected its downstream signal transduction.Besides, after incubated with FOXM1 si RNA and lnc RNA-PVT1 plasmid in TGF-β1 treated MRC-5 cells,the expression levels of fibroblast activation markers were decreased with the down-regulation of FOXM1,rescued TGF-β1-induced the activation of MRC-5 cells.However,the up-regulation of lnc RNA-PVT1 reversed this effect.Together,these results indicate that FOXM1 could act as a profibrotic factor in pulmonary fibroblasts by elevating lnc RNA-PVT1.Conclusion In the present study,we discover that lnc RNA-PVT1 plays a key role in the activation of lung fibroblasts,and confirm that lnc RNA-PVT1 not only competitively binds mi R-497-5p to influence the expression of Smad3 and Bcl2,but also is positively regulated by the transcription factor FOXM1,forming a FOXM1/lnc RNA-PVT1/mi R-497-5p axis in the activation of lung fibroblasts during silica-induced pulmonary fibrosis.Our research may provide potential biological targets for the prevention and treatment of silicosis.