| Background and objectiveIdiopathic pulmonary fibrosis(IPF)is a chronic and progressive fibrotic lung disease of unknown cause.Usual interstitial pneumonia(UIP)is the main histopathologic and radiologic pattern.IPF causes honeycombing,irreversible tissue damage,and respiratory failure,which would seriously affect the life quality and increase the mortality rate.IPF,with a high incidence,accounts for around 20%of all cases of interstitial lung diseases.It occurs more commonly among old adults.Males would be affected more often than females.Globally,around 3 million people suffer from it.IPF is easy to be misdiagnosed while the pathogenesis is extremely complicated and.Lacking effective drug treatments,IPF has an extremely poor prognosis with a median survival time of 3-5 years if the suffered patients do not undergo transplantation.Therefore,researching highly specific biomarkers and investigating the mechanism of new targets are of great significance for the diagnosis and prevention of IPF.Circular RNA(circular RNA,circRNA)is a new type of non-coding RNA.They are connected end-to-end during the transcription forming covalently-closed loop structures and play a key role in a variety of pathophysiological processes.Most circRNAs are highly abundant,stable,and conservative among species while often show specific expression patterns among different tissues.Recently,as a hot research topic,it has been found that functional circRNAs participated in the regulatory network regulating gene expression at the transcriptional and post-transcriptional levels,which could affect the occurrence and progression of various diseases such as pulmonary fibrosis and lung cancer through competitive inhibition of microRNAs and other mechanisms.Yet,despite progress in the research of circRNAs,their mechanism in IPF is still at the early exploration stage with more novel functional circRNAs need to be studied since their mechanism in the regulatory network is not completely clear.Here,we constructed a mouse model for IPF and analyzed the expression difference of circRNAs between this model and control aminals by the high-throughput sequencing method.We found mmu＿circ＿0001699 had the most significant expression difference among these circRNAs tested in this model,suggesting it may play an important role in the occurrence and development of IPF.In the current study,we aimed to initially explore the role of mmu＿circ＿0001699 in the occurrence and development of IPF,which would provide a useful example as a scientific experimental foundation for the future study of other circRNAs that might become diagnostic biomarkers and potential molecular therapeutic targets for IPF.This research content has three parts:1)the analysis of the difference of circRNAs in the bleomycin-induced mouse model of IPF;2)the regulation of mmu＿circ＿0001699 on the activation,proliferation,and migration of lung fibroblasts;3)investigation on the mechanism of mmu＿circ＿0001699 in the occurrence and development of IPF.Part Ⅰ Difference analysis of circRNAs in bleomycin-induced mouse idiopathic pulmonary fibrosis modelMethods1.To construct a BLM-induced mouse idiopathic pulmonary fibrosis(BLM-IPF)model,C57BL/6J male mice with a weight between 22-26g and age of 6-8 weeks were selected,anesthetized intraperitoneal injection of 1%sodium Pentobarbital,instilled with bleomycin(BLM)solution(2.5 u/kg)into the lung through a laryngoscope,and then routinely fed for 28 days.2.Collect BLM-IPF lung tissue specimens,observe the pulmonary fibrosis lesions by pathological section,HE and Masson staining,determine the content of hydroxyproline,and detect ColI,fibronectin(fibronectin,Fn),and α-SMA their expression level by immunofluorescence and qRT-PCR.,to verify the success of the model building.3.Select 3 pairs of IPF lung tissue and normal lung tissue specimens for high-throughput second-generation sequencing;Using biosynthesis analysis software,perform enrichment analysis for the circRNAs’ parental genes expression difference based on Gene Ontology and KEGG,and correlation analysis between circRNA and miRNA.4.The qRT-PCR method detects the expression of circ＿0001699,circ＿0001084,circ＿0000368,and circ＿0000831 in the lung tissues of 15 pairs of BLM-IPF mice and normal lung tissues of mice.5.Divergent primer amplification,cloning and sequencing to verify the existence of circular mmu＿circ＿0001699 in the IPF tissue samples.Results1.Bleomycin solution was instilled through the tracheal intubation cannula to obtain lung tissues of BLM-IPF mice.After pathological sectioning,the typical pathological manifestations of pulmonary fibrosis were observed by HE and Masson staining;compared to the control group,in the model mice,Hydroxyproline level was significantly increased(P<0.05);the expression levels of ColI,Fn,and a-SMA expression level detection in model mice were significantly higher than those in the control group by qRT-PCR and immunofluorescence(P<0.05).2.High-throughput sequencing technology detection and analysis of 3 pairs of lung tissue specimens,there are 7168 circular RNA transcripts.In the IPF group,there are 240 up-regulated circRNAs and 279 down-regulated circRNAs;biosynthesis analysis shows that the expression of 60 circRNAs showed significant difference between the 2 groups(fold change>2.0,P<0.05).3.Gene Ontology analysis for the circRNAs’ parental genes expression difference showed that differentially expressed circular RNAs might be involved in the retrograde transport to the Golgi body and the development of dendritic spines.They also affected the production and regulation of miRNAs involving in gene silencing through miRNAs as well as reaction to reactive oxygen species,synaptic fusion protein binding,positive regulation of stress fiber components,and other biological functions.KEGG analysis shows that they might be involved in the TGF-p signaling pathway,cell cycle,focal adhesion,bacterial invasion through the epithelium,autophagy,regulation of signal pathways such as transcription disorders in cancer.The correlation analysis between circRNA and miRNA predicted the four most differentially expressed miRNAs were regulated by circRNA.4.Compared with normal mouse lung tissue,the relative expression levels of circ＿0001699 and circ＿0001084 in the BLM-IPF group were significantly increased,while the relative expression levels of circ＿0000368 and circ＿0000831 were significantly decreased(P<0.05),which was consistent with the high-throughput sequencing results.mmu＿circ＿0001699 was selected for the next experiment due to the most significant difference changes.5.Sanger sequencing results showed that it was completely consistent with the mmu＿circ＿0001699 sequence in circBase,and there was a BS site connected end to end in the second exon of Smadl,indicating that there was a circular mmu＿circ＿0001699 in the IPF tissue samples.Part Ⅱ The regulatory effect of mmu＿circ＿0001699 on the activation,proliferation and migration of lung fibroblastsMethods1.Isolate,culture and identify primary lung fibroblasts from C57BL/6J mice within 3 days of birth through primary culture technology,and induce primary lung fibroblasts with 10ng/mL TGF-β1(+/-)for 48 h.The morphological changes were observed under the microscope,and the relative expression levels of ColI,Fn and α-SMA were detected by qRT-PCR after TGF-β1(+/-)induced primary lung fibroblasts.qRT-PCR was used to detect the expression of mmu＿circ＿0001699 in lung fibroblasts.2.Divergent primer RT-PCR amplification,molecular cloning and Sanger sequencing to verify the existence of circular mmu＿circ＿0001699 in primary fibroblasts.3.Design and synthesize siRNA that specifically down-regulate the expression of mmu＿circ＿0001699 and unrelated sequences,transfect them into primary mouse lung fibroblasts with lipofectamineTM3000 for 6-8 hours,and then induce the cells 48 hours by TGF-β1.qRT-PCR detected the relative expression levels of mmu＿circ＿0001699 in the three groups of cells(si-circ＿0001699 group,si-NC group,and Blank group).4.qRT-CR detects the expression of α-SMA,ColI and fibronectin in three groups of cells(si-circ＿0001699 group,si-NC group,and Blank group).5.Four groups of primary lung fibroblasts were detected by EDU staining and MTT test for their proliferation function(TGF-β1 group,TGF-β1+si-NC group,TGF-β1+si-circRNA group and Control group.The first three groups were all treated by TGF-β1 induction,control group was not induced by TGF-β1).6.By using the scratch test and Transwell test to detect the migration ability of four groups of primary lung fibroblasts(TGF-β1 group,TGF-β1+si-NC group,TGF-β1+si-circRNA group and Control group).7.The mmu＿circ＿0001699 shRNA packaged with adeno-associated virus(AAV)was instilled intratracheally into 5-week-old C57BL/6 J male mice at the level about 30μL(2.0×1013 viral particles/mL),then they were continued fed for 3 Weeks,they were fed normally for 28 days after giving bleomycin solution.Masson staining was used to observe pulmonary fibrosis.qRT-PCR was used to detect the expression of ColI,Fn,and a-SMA in the tissue;immunofluorescence was used to detect the relative expression of a-SMA and Ki67 in lung tissue samples.Results1.Isolate,culture and identify mouse primary lung fibroblasts through primary culture technology.Typical fibroblast morphology was seen under the microscope.Lung fibroblasts were induced with lOng/mL TGF-β1 to promote their abnormal activation and transformation into myofibroblasts.At the same time,it mediated the collagen synthesis and adhesion process of the fibroblasts.qRT-PCR detected the lung tissue after TGF-β1 induction their expressions of ColI,Fn and α-SMA in fibroblasts were significantly higher than those in the control group(P<0.05).Compared with the control group,TGF-β1 induced a significant increase of the expression level of mmu＿circ＿0001699 in primary lung fibroblasts(P<0.05).2.Sanger sequencing confirmed that there was a BS site connected end to end in the second exon of Smadl in primary lung fibroblasts,indicating that there is a circular mmu＿circ＿0001699 in the cells.3.After down-regulating mmu＿circ＿0001699,qRT-PCR test results showed that the expression level of mmu＿circ＿0001699 in the primary lung fibroblast group was significantly lower than that of the control group(P<0.05);qRT-PCR results showed that the expression of ColI,Fn and a-SMA was significantly reduced(P<0.05),suggesting that downregulation of mmu＿circ＿0001699 can inhibit the abnormal transformation of fibroblasts into myofibroblasts,collagen formation and adhesion.4.EDU experiment results showed that down-regulating mmu＿circ＿0001699 significantly reduced the proliferation of fibroblasts composed of si＿circ＿0001699(P<0.05);MTT experiment results showed that down-regulating mmu＿circ＿0001699 significantly reduced the viability of fibroblasts induced by TGF-β1(P<0.05).It suggests that mmu＿circ＿0001699 is involved in regulating the proliferation of fibroblasts induced by TGF-β1.5.The results of Transwell experiment showed that down-regulation of mmu＿circ＿0001699 reduced the number of fibroblast transmembrane cells induced by TGF-β1(P<0.05);the scratch area calculated by Image J software showed that the scratch healing rate of the si＿circ＿0001699 group was significantly lower than that of the control group.(P<0.05).It suggests that down-regulation of mmu＿circ＿0001699 can inhibit the migration function of fibroblasts.6.AAV-related mmu＿circ＿0001699 shRNA was instilled into the trachea of mice,and then bleomycin solution was instilled.Masson staining showed that lung fibrosis was significantly reduced compared with the control group;qRT-PCR detected the expression of ColI,Fn,α-SMA was significantly lower than that of the control group;immunofluorescence showed that the expression of myofibroblast marker(a-SMA)was reduced,P<0.05,the difference was significantly different.The results suggest that inhibiting mmu＿circ＿0001699 in vivo can significantly reduce the occurrence and development of bleomycin-induced idiopathic pulmonary fibrosis.Part Ⅲ Investigation on the mechanism of mmu＿circ＿0001699 in the occurrence and development of idiopathic pulmonary fibrosisMethods1.Bioinformatics technology analyzes mmu＿circ＿0001699 sequence and predicts the miRNA related to it.The dual luciferase reporter experiment and the RNA binding protein immunoprecipitation experiment(RIP)confirmed that mmu＿circ＿0001699 binded to miR-98-5p.qRT-PCR detection down-regulated the expression level of miR-98-5p in primary lung fibroblasts after mmu＿circ＿0001699.2.Bioinformatics technology analysis miR-98-5p has a targeting relationship with HMGA2 and Edn1.The dual luciferase report experiment confirmed that HMGA2 and Ednl were the target genes of miR-98-5p.Western blot was used to detect the effect of up-regulation of miR-98-5p on the expression of HMGA2 and Ednl in fibroblasts.3.Up-regulation of miR-98-5p expression,qRT-PCR was used to detect the expression level of myofibroblast specific marker a-SMA,immunofluorescence to detect the average fluorescence intensity of a-SMA in cells,MTT experiment to detect lung fibrosis the effect of cell proliferation and Transwell was used to detect the effect of lung fibroblast migration.4.Response experiment:use HMGA2 and Ednl recombinant vectors pcDNA3.1-HMGA2 and pcDNA3.1-Ednl without 3’UTR region,and transfect them with mmu＿circ＿0001699 siRNA or miR-98-5p mimics alone or together to Lung fibroblasts.The effect of relative fluorescence intensity response of a-SMA in cells on abnormal activation of fibroblasts was detected by immunofluorescence;changes in cell proliferation ability were detected by MTT experiment;changes in cell migration ability were detected by Transwell experiment.Results1.Bioinformatics predictive analysis showed that mmu＿circ＿0001699 and miR-98-5p had complementary regions that bonded to each other.The results of the dual luciferase report experiment showed that the luciferase activity values of the miR-98-5p mimics and WT＿circRNA＿pmirGLO groups were significantly lower than the other three groups(P<0.05);RIP results showed that in the Ago2 co-precipitation product,the abundance of mmu＿circ＿0001699 and miR-98-5p was significantly higher than that of the IgG negative control group(P<0.05),indicating that mmu＿circ＿0001699 can specifically bind to miR-98-5p.After down-regulating mmu＿circ＿0001699,the expression level of miR-98-5p in primary lung fibroblasts induced by TGF-β1 was significantly higher than that in the control group(P<0.05),suggesting that mmu＿circ＿0001699 also has a negative regulatory effect on miR-98-5p.2.Bioinformatics analysis results showed that the 3’UTR regions of HMGA2 and Ednl had complementary regions that interact with miR-98-5p;the dual luciferase report showed that when miR-98-5p mimics were co-existing with WT＿HMGA2＿pmirGLO or WT＿Edn1＿pmirGLO,the luciferase activity of the transfection group was significantly lower than that of the control group(P<0.05);Western blot was used to detect the expression of HMGA2 and Ednl in miR-98-5p lung fibroblasts were significantly decreased(P<0.05)than the other two groups(TGF-β1 group,TGF-β1+miRNA scramble group),indicating that miR-98-5p can target and negatively regulate HMGA2 and Ednl.3.After up-regulating miR-98-5p,the results of qRT-PCR and immunofluorescence detection showed that the relative expression of a-SMA and the average fluorescence intensity decreased significantly(P<0.05);the results of MTT experiment showed that up-regulating miR-98-5p Significantly reduced the proliferation ability of lung fibroblasts(P<0.05);Transwell experiment results showed that the number of transmembrane cells in the miR-98-5p mimics group was significantly lower than that in the control group(P<0.05).The results suggest that up-regulation of miR-98-5p can inhibit the abnormal transformation of fibroblasts into myofibroblasts,inhibit the proliferation and migration of fibroblasts.Combining with the results of the second part,this suggests that down-regulation of mmu＿circ＿0001699 and up-regulation of miR-98-5p can affect lung fibrosis.The cell effect is the same.4.Response experiment:pcDNA3.1-HMGA2 and pcDNA3.1-Ednl overexpression vectors co-transfected with circ＿0001699 siRNA or miR-98-5p mimics reduced the primary lung formation of circ＿0001699 siRNA or miR-98-5p mimics inhibition of abnormal activation,proliferation and migration of fibroblasts,the difference was statistically significant(P<0.05).The results showed that miR-98-5p acted on HMGA2 and Ednl without 3’UTR,suggesting that HMGA2 and Ednl play a regulatory role downstream of miR-98-5p.Conclusions1.Many circRNAs has differential expression in bleomycin induced idiopathic pulmonary fibrosis mouse model,wherein mmu＿circ＿0001699 highly expressed in idiopathic pulmonary fibrosis tissue sample.2.Down-regulating the expression of mmu＿circ＿0001699 can inhibit the abnormal activation,proliferation and migration of lung fibroblasts,and can reduce the degree of pulmonary fibrosis.3.In the occurrence and development of idiopathic pulmonary fibrosis,mmu＿circ＿0001699 can participate in regulating the activation,proliferation and migration of lung fibroblasts through the miR-98-5p/HMGA2&Ednl axis,and is expected to become a new diagnostic biomarker and potential therapeutic target. |
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