| Radiation-induced pulmonary fibrosis（RIPF）is a common and refractory complication of radiation therapy for thoracic tumors and in the late phase of nuclear radiation emergencies,which is progressive and irreversible.RIPF may initially present as pneumonia,and progressively to chronic debilitating fibrosis in severe cases,characterized by loss of alveolar structure,disordered thickening of septum,collapse of alveolar interstitial,and replacement of normal parenchyma with fibrous tissue.Radiation can lead to extracellular matrix（ECM）deposition,apoptosis,lung cell senescence,and loss of barrier function,resulting in increased inflammatory response,leukocyte infiltration,and production and secretion of cytokines,chemokines,and growth factors,contributing to chronic hypoxia.RNA function is associated with a variety of methylation modifications,of which,m6A is the most abundant and widespread RNA modification in epitranscriptomics.The m6A modification is dynamically reversible and plays a dynamic regulatory role in the context of physiological changes in the organism,mainly in biological processes such as DNA damage response,regulation of fibrotic progression,and tumorigenesis.Due to the lack of effective treatments and the uncertainty of the latency period for RIPF,its mortality rate is higher than that of many cancers.Therefore,in-depth understanding of the pathogenesis of radiation lung injury,searching for ideal molecular targets,and elucidating the mechanism of epigenetic modification,we aimed to provide new therapeutic targets and experimental basis for the prevention and treatment of RIPF.Research Background:GATA binding protein-3（GATA3）is a transcription factor that regulates target genes and triggers the proliferation and differentiation of the specific immune cells.GATA3 plays a key role in the development of Th2 phenotype,and the overexpression of GATA3 exacerbates the development of pulmonary fibrosis by inducing a shift in the Th1/Th2 balance to a Th2 phenotype.m6A,an epigenetic modification of RNA molecules,regulates RNA splicing,translocation,and stability,which in turn affects protein synthesis and important biological processes,such as DNA damage response,lung damage,and tumor development,in mammals.It has been demonstrated that overexpression of GATA3 plays a key role in regulating the process of pulmonary fibrosis by inducing the conversion of Th1/Th2balance to Th2,and that Th2 cells can promote fibroblast proliferation and collagen production through upregulating cytokines IL-4 and IL-13.Moreover,GATA3 can affect the migratory and invasive ability of cells through m6A modification.However,most recent studies of m6A modification of GATA3 focus on tumor immunity,and the mechanism of GATA3 in RIPF is rarely reported.Research Purposes:Through studying the relationship between VIRMA and GATA3 mRNA m6A modification,and the molecular mechanism of GATA3 activating the TGF-β/Smads signaling pathway to induce EMT and ECM deposition through the transcriptional regulation of NRP1,we aim to provide new therapeutic targets and experimental bases for the prevention and treatment of radiotherapy complications in clinical thoracic malignancies.Research Methods:First,in this study,a mouse model of RIPF was established by localized irradiation of the mouse thorax with a single X-ray irradiation of 20 Gy（with a dose rate of 2.0Gy/min）,and the mouse model of RIPF was characterized by detecting hydroxyproline content using H&E staining,Masson staining and alkaline hydrolysis.A human lung epithelial cell line（A549）and a mouse lung epithelial cell line（MLE-12）was received a single X-ray irradiation,respectively,and establish the radiation-induced pulmonary fibrosis phenotypic epithelial cell model,and using q RT-PCR and Western Blot experiments to detecting of Collagen-1 andα-SMA expression to characterize the radiation induced fibrosiss phenotype of lung epithelial cells.In line with,the human endothelial cells（HVUEC）and human fibroblastscells（HFL-1）were chosen as compared group,they were also received a single X-ray irradiation.Furthermore,total m6A level was quantified by a colorimetric assay.The expression changes of m6A modifying enzymes,VIRMA,YTHDF1 and FTO,were detected using q RT-PCR and Western Blot experiments.The m6A modified differential expression genes were detected by comparing control and irradiated cells using Me RIP-Seq assay.The cell models of knockdown or overexpressing VIRMA and YTHDF1were generated by lentiviral infection method,respectively.The protein expression levels of VIRMA,YTHDF1,E-cadherin,Vimentin,N-cadherin,α-SMA and Collagen-1 were detected using Western Blot experiment.The gene expression levels of VIRMA,YTHDF1,CDH1,CDH2,VIM,ACTA2 and COL1A1 were detected using q RT-PCR.The enrichment of m6A on GATA3 mRNA was detected using Me RIP-qPCR in cell models.Using RIP-qPCR to check VIRMA or YTHDF1 proteins bind to GATA3mRNA.A dual luciferase reporter gene assay was performed to identify the regulatory relationship of VIRMA and GATA3.Then,the regulatory effect of VIRMA on GATA3mRNA stability was detected using a RNA synthesis inhibitor Act D.The regulatory effect of YTHDF1 on GATA3 protein stability was detected using a protein synthesis inhibitor CHX.Through above experiments,we aim to explore the role of m6A modification in the development of radiological lung fibrosis.Finally,GATA3 or NRP1 knockdown or overexpression cell models were established to explore the regulatory relationship between GATA3 and NRP1.The binding sites of GATA3 and NRP1 promoter region were predicted by transcription factor bioinformatics website,and the binding of GATA3 to NRP1 promoter region was identified by Ch IP-qPCR.The regulatory relationship of GATA3 to NRP1 was detected using a dual luciferase reporter gene assay;an immunofluorescence staining assay was performed to check the entrance of GATA3 into cell nucleus..NRP1 binds to TGF-βprotein was detected using an immunoprecipitation assay.the expression levels of GATA3 and NRP1 protein was detected by IHC assay in lung tissues.RT-qPCR was used to detect the expression levels of GATA3,NRP1,CDH1,CDH2,VIM,ACTA2,and COL1A1 mRNA in cells.The expression levels of GATA3,NRP1,E-cadherin,N-cadherin,Vimentin,α-SMA,Collagen-1 and TGF-β/Smad pathway-related proteins were detected in the cells using Western blot.Through above experiments,we aim to further explore the molecular mechanism of GATA3 in the development of radiological pulmonary fibrosis.Research Results:1.Changes in total m6A levels during the formation of radiation-induced pulmonary fibrosis（1）In this study,we used a single high-dose X-ray irradiation to establish an in vivo RIPF mouse model.H&E staining showed that the alveolar walls of the control mice were slender,the structure was clearly visible,and the capillaries were intact.The abnormal structure of alveolar tissue was observed in RIPF group,liking the thickening of the texture in lung tissues,a small number of alveolar atrophy and collapse,and a small amount of inflammatory cell infiltration,4 and 8 weeks after irradiation.Sixteen weeks after irradiation,the peripheral alveoli fused with each other and dilated,and the cell proliferated obviously around the blood vessels.Twenty-four weeks after irradiation,a large increase in interstitial cells,fibrous tissue hyperplasia,and obvious lung solidification were observed.（2）Masson staining showed that in control mice,the alveolar structure was clear and no obvious fibrous hyperplasia were observed,the fibrous staining was mainly concentrated around the blood vessels,and the staining was light.Eight and sixteen weeks after irradiation,the alveolar structure was obviously damaged,and blue collagen deposition began to appear.With the prolongation of time,obvious hyperplasia of fibrous tissue can be seen in the alveolar septum and alveolar lumen at 24 weeks after irradiation,presenting the bundles of blue color and obvious collagen deposition.The surface of the lung tissue in the control group was smooth and bright red in color,and there was no obvious abnormality.However,the lung tissues of mice were observed as dark red color 24 weeks after irradiation,showing obvious signs of pulmonary fibrosis,such as inflammation and grayish-white areas of dense solid changes.（3）The results of hydroxyproline（HYP）content assay showed that the HYP content gradually increased content of HYP along with the extension of time after irradiation,and the HYP content was the highest at 24 weeks.Further results of IHC,Western Blot and q RT-PCR showed that,compared with the control group,the expression ofα-SMA and Collagen-1 proteins increased significantly with time after irradiation and reached the highest at 24 weeks after irradiation.In consistent with the trend of protein results,mRNA levels also reached the highest at 24 weeks after irradiation,ACTA2 elevated to 5.16-fold（P<0.01）and COL1A1 elevated to 22.68-fold（P<0.01）compared with control.In conclusion,the mouse model of radiation-induced pulmonary fibrosis was successfully constructed.（4）Changes in total m6A levels in the lung group of RIPF mice showed that compared with the control group,the total m6A levels in the lung tissues of irradiated mice gradually increased along with time,and reached the highest at 24 weeks after irradiation（P<0.01）.Western Blot and q RT-PCR assays were used to detect the changes in protein and mRNA expression of m6A-modifying enzymes,VIRMA,FTO and YTHDF1,in the lungs of RIPF mice.Compared with the control group,the protein and mRNA expression of the methylase VIRMA and the recognition protein YTHDF1 were significantly elevated with the extension of irradiation over time（P<0.05）,while the protein and mRNA expression of demethylase FTO was significantly reduced（P<0.05）,.It suggested that the changes in m6A modification accompany the formation of radiolucent pulmonary fibrosis in mice.2.Changes in total m6A levels during radiation-induced fibrotic phenotype formation in lung cells（1）First,human alveolar epithelial cells A549,mouse lung epithelial cells MLE-12,human lung endothelial cells HVUEC and human lung fibroblasts HFL-1 were subjected to a single X-ray irradiation to construct radiation-induced pulmonary fibrosis phenotypic epithelial,endothelial and fibroblast cell lines.The results of total m6A levels showed that total m6A levels were significantly higher in the irradiated cell groups,epithelial cell lines（A549 and MLE-12）and endothelial cell lines（HVUEC）,and lower in the irradiated group of fibroblast cell line（HFL-1）compared with the control group.The results of Western Blot and q RT-PCR showed that,as compared with the control group,the VIRMA protein and mRNA expression was significantly increased in the irradiated A549,MLE-12 and HVUEC cells（P<0.05）and decreased in the irradiated HFL-1 cells（P<0.05）;GATA3 protein and mRNA expression was significantly increased in the irradiated A549 and MLE-12 cells（P<0.05）and decreased in the irradiated HVUEC and HFL-1 cells（P<0.05）;while NRP1 protein and mRNA expression was significantly elevated in the irradiated A549,MLE-12,HVUEC and HFL-1 cells（P<0.05）.（2）The results of Western Blot and q RT-PCR showed significantly higher levels ofα-SMA and Collagen-1 protein and mRNA expression in the irradiated group than control group（P<0.05）.It indicated that the radiation-induced fibrosis phenotype of lung epithelial cells was constructed successfully.（3）The results using Western Blot and q RT-PCR showed that the protein and mRNA expression of methylase VIRMA and recognition protein YTHDF1 were significantly increased（P<0.01）,and the protein and mRNA expression of demethylase FTO were significantly decreased（P<0.01）in irradiated cells compared with the control group.Immunofluorescence results showed that the protein expression of methylase VIRMA was significantly elevated in the irradiated cells compared with the control group.It suggestes that the formation of radiation-induced fibrotic phenotype in lung epithelial cells is accompanied by changes in m6A modification,and that methylase VIRMA is positively correlated with the process of lung fibrosis.3.To explore the molecular mechanism of m6A modification in regulating radiation-induced pulmonary fibrosis（1）In order to elucidate the mechanism of methylase VIRMA-mediated m6A modification in regulating the formation of RIPF,lentiviral infection was utilized to construct cell models with knockdown and overexpression of the VIRMA.The changes of total m6A levels in these cell models were detected by colorimetric quantification,and the results showed that in A549 and MLE-12 cells knocking down VIRMA,the total m6A levels were significantly reduced（P<0.01）.In contrast,the total m6A levelin VIRMA overexpressed cells was significantly increased（P<0.01）.Changes in the expression of fibros actin（F-actin）in cells were detected using immunofluorescence,and the results showed that compared with the control group,the membrane region and perinuclear region of irradiated cells presented a obvious stress fiber network of cytoskeletal proteins（F-actin）,and after the VIRMA gene was knocked down,there were some short,scattered and unorganized fiber networks only in the membrane region.organized fiber networks.Since F-actin is associated with epithelial-mesenchymal transition（EMT）,this suggests that interfering with the expression of the VIRMA gene inhibits radiation-induced EMT occurrence and further affects cell morphology.Western Blot and q RT-PCR were used to detect the changes in the expression of EMT markers and fibrosis markers after knocking down the VIRMA gene in the cells,and the results showed that compared with NC,the protein and mRNA expressions of the epithelial markers E-cadherin were significantly reduced after irradiation,and the mesenchymal markers N-cadherin and Vimentin,as well as the fibrosis markersα-SMA and Collagen-1 protein and mRNA expression were significantly elevated（P<0.01）.In contrast,compared with irradiation alone,in the VIRMA knockdown cells concurrent administrated with irradiation,E-cadherin protein and mRNA expression was elevated,while N-cadherin,Vimentin,α-SMA,and Collagen-1 protein and mRNA expression were significantly reduced（P<0.01）.This suggests that interfering with the expression of VIRMA gene can inhibit radiation-induced EMT occurrence and fibrosis process.The changes in the expression of EMT markers and fibrosis markers afteroverexpression of VIRMAwere further detected,and the results showed that compared with Vector,the E-cadherin protein and mRNA expression was significantly decreased in the overexpression of VIRMA group,but the protein and mRNA expression of N-cadherin,Vimentin,α-SMA and Collagen-1 was significantly elevated（P<0.01）.Taken together,this suggests that the methylase VIRMA regulates the onset and development of fibrosis by affecting the level of m6A modification.（2）After the positive correlation between RIPF formation was positively and m6A modification was determined,to further elucidate the molecular mechanism of m6A modification in the formation of RIPF,epitranscriptome Me RIP-seq analysis was performed using the X-ray irradiated cells.The results revealed that comparing the abundance of 6,206 m6A peaks differed between the control cells and irradiated cells,701 hypermethylated m6A peaks（Fold Change≥2）with 331 genes significantly up-regulated at their mRNA levels,which are referred to as hypermethylated genes,and370 genes down-regulated at their mRNA levels.804 hypomethylated m6A peaks were also identified,with 349 genes significantly upregulated and 455 genes downregulated at the mRNA level.GO analysis of these genes undergoing hypermethylation showed that these genes were mainly associated with RNA-specific processes,including RNA catabolic processes,RNA metabolic processes,RNA transport,RNA localization and translation.KEGG enrichment analysis of genes undergoing hypermethylation showed that the functions of these genes were mainly enriched in the regulating the actin cytoskeleton,focal adhesion,adhesion junction,ECM-receptor interaction,and Focal adhesion pathway.By analyzing RIPF-Me RIP-seq and RIPF-RNA-seq data,the genes that were hypermethylated and up-regulated were intersected with the downstream target genes of VIRMA,and a total of one highly expressed and hypermethylated m6A-modified gene was screened,namely GATA3.The m6A enrichment of GATA3 mRNA in the radiation-induced fibrotic phenotype of lung epithelial cells was detected by Me RIP-qPCR assay.The results showed that the m6A enrichment of GATA3 mRNA in irradiated cells was significantly increased compared with that in control group（P<0.01）.Visualization analysis showed the significant m6A enrichment in the 3’-UTR region of GATA3 mRNA,with 64.7-fold enrichment.To further elucidate the role of m6A modification in regulating GATA3expression,we performed a dual luciferase reporter gene assay using two different luciferase reporter gene plasmids,a wild-type 3’-UTR region of GATA3 mRNA（pmir GLO-GATA3-WT）and a mutant plasmid.pmir GLO-GATA3-MUT,with replacing adenosine（A）bases in the 3’-UTR region of GATA3（chr10:8074332-8074512）with cytosine（C）bases instead.The results showed that the luciferase activity of pmir GLO-GATA3-WT increased significantly after VIRMA overexpression,whereas pmir GLO-GATA3-MUT did not show any significant changes after VIRMA overexpression,which suggesting that VIRMA-mediated m6A modification on the GATA3 3’-UTR regulates the GATA3 expression changes.The binding of VIRMA protein to GATA3 mRNA in the cells was detected by RIP-qPCR assay,the results showed that a significantly increased binding between VIRMA protein and GATA3mRNA after providing irradiation to the cells.The m6A enrichment of GATA3 mRNA in VIRMA knockdown cells was detected by Me RIP-qPCR assay,and the results showed that the m6A enrichment of GATA3 mRNA was significantly reduced after deletion of VIRMA gene（P<0.01）.Taken together,we inferred that GATA3 acts as a direct downstream target gene for VIRMA-mediated m6A methylation.The changes in GATA3 protein and mRNA expression after knocking down VIRMA gene in cells were detected using Western Blot and q RT-PCR.The results showed that GATA3 protein and mRNA expression was significantly higher after irradiation compared with NC（P<0.01）,and GATA3 protein and mRNA expression in cells of the group that were knocked down the VIRMA gene combined with irradiation was significantly decreased（P<0.01）.In contrast,changes in GATA3 protein and mRNA expression were significantly elevated after overexpression of VIRMA gene（P<0.01）.The mRNA decay was induced by treating the cells with knocking down VIRMA gene using 5 mg/m L Act D.The changes of GATA3 mRNA expression were detected with the prolongation of the treatment time.The results showed that the half-life of GATA3 mRNA was prolonged in the irradiation-only group compared with the NC group,whereas knocking down the expression of VIRMA gene reduced the expression of GATA3 mRNA and shortened the half-life of GATA3 mRNA.In summary,VIRMA protein inhibits GATA3 mRNA degradation in an m6A-dependent manner,thereby regulating GATA3 expression.（3）In order to further elucidate the m6A modification mechanism of GATA3,lentiviral infection was utilized to construct cell models for knockdown and overexpression of YTHDF1 gene.The binding between YTHDF1 protein and GATA3mRNA in the cells was detected by RIP-qPCR assay,and the results showed that the binding between YTHDF1 protein and GATA3 mRNA in the irradiated group was significantly increased（P<0.05）.Changes in total m6A levels in cells knocked down YTHDF1 gene were detected using colorimetric quantification,and the results showed that the total m6A levels in cells knocked down YTHDF1 gene were significantly reduced（P<0.01）.The changes of GATA3 protein and mRNA expression after knocking down YTHDF1 gene in two RIPF cell models were detected by using Western Blot and q RT-PCR,and the results showed that the expression of GATA3 protein and mRNA in the knocked-down YTHDF1 cell group was significantly reduced（P<0.01）.In contrast,when overexpressing YTHDF1 gene,the total cellular m6A level was significantly increased（P<0.01）.GATA3 protein and mRNA expression was significantly higher in the overexpressed YTHDF1 group compared to Vector（P<0.01）.Using protein stability assay,cells knocking down YTHDF1 gene were treated with 10μM CHX to induce protein decay,and changes in GATA3 protein expression were detected with the extension of treatment time.The results showed that the half-life of GATA3 protein translation was prolonged in the irradiation alone group.Compared with the irradiation alone group,while knockdown of YTHDF1 gene reduced the expression of GATA3 protein and shortened the half-life of GATA3 protein translation.In summary,the recognition protein YTHDF1 inhibits the degradation of GATA3protein in an m6A-dependent manner,thus regulating the expression of GATA3.4.To explore the molecular mechanism of transcription factor GATA3 in radiation-induced pulmonary fibrosis（1）First,the correlation between GATA3 and RIPF process was verified.Western blot and q RT-PCR results showed that,the protein expression of GATA3 increased with the extension of time after irradiation,and reached the highest at 24 weeks after irradiation,and the mRNA level increased to 1.99-fold（P<0.01）.IHC results showed that compared with the control group,the brown GATA3-positive cells in lung tissue gradually increased with the extension of time after irradiation,and reached the highest at 24 weeks after irradiation,and the positive cells were almost covered in the lung tissue.IHC results showed that,brown GATA3-positive cells gradually increased in the lung tissue with the extension of time,and reached the highest at 24 weeks after irradiation,and the lung tissue was almost covered with positive cells.Western Blot and q RT-PCR results of radiation-induced fibrotic phenotype in lung epithelial cells showed that the expression of GATA3 protein and mRNA in cells was significantly increased after irradiation,and in A549 cells,the GATA3 increased to 2.37-fold after irradiation（P<0.01）.In MLE-12 cells,GATA3 was elevated to 1.33-fold（P<0.01）.It suggestes that the elevation of GATA3 expression is related to the process of radiological pulmonary fibrosis.（2）In vitro experiments confirmed the cytological mechanism by which the GATA3 gene regulates radiographic pulmonary fibrosis.A cellular model of knockdown and overexpression of GATA3 gene was constructed using lentiviral infection.After targeting and inhibiting GATA3 in cells,changes in the expression of EMT markers and fibrosis markers were detected using Western Blot and q RT-PCR.The results showed that the expression of the epithelial marker E-cadherin protein and mRNA was significantly reduced after irradiation,and the mesenchymal markers N-cadherin and Vimentin,as well as fibrosis markers alpha-SMA and Collagen-1 protein and mRNA expression were significantly elevated（P<0.05）.In contrast,knockdown of GATA3 gene while giving irradiation group cells had elevated E-cadherin protein and mRNA expression,and N-cadherin,Vimentin,α-SMA and Collagen-1 protein and mRNA expression were significantly decreased（P<0.05）.Further testing after overexpression of the GATA3 gene showed that the epithelial marker E-cadherin protein and mRNA expression was significantly lower,and the mesenchymal markers N-cadherin and Vimentin,as well as the fibrosis markersα-SMA and Collagen-1protein and mRNA expression were significantly higher in the overexpression of VIRMA group（P<0.01）.Taken together,this suggests that the transcription factor GATA3 is involved in the initiation and development of fibrosis.（3）The molecular mechanism of NRP1 regulation by transcription factor GATA3was analyzed by GO analysis of the changed genes in RIPF-RNA-seq,and the changed genes were mainly associated with mainly with transcriptional activity.KEGG enrichment analysis showed that the changed genes were mainly enriched in the TGF-beta signaling pathway.By analyzing the idiopathic pulmonary fibrosis dataset GSE24206 and RIPF-RNA-seq in the GEO database,11 genes were screened out by taking the intersection of the changed genes with the downstream target genes of the transcription factor GATA3 and the proteins related to the TGF-beta signaling pathway.The expression changes of these 11 genes in cells given X-ray irradiation were detected by q RT-PCR,and the most significant change in NRP1 expression was elevated to 2.32-fold（P<0.01）.Correlation analysis was performed using the GEO database,and NRP1 showed a significant correlation（R=0.456）.A cell model for knockdown of NRP1 gene was constructed using lentiviral infection.The results showed that NRP1 protein and mRNA expression was significantly elevated after irradiation compared with NC（P<0.01）.Compared with irradiation alone,knocking down GATA3 gene while giving irradiation group cells NRP1 protein and mRNA expression were significantly decreased（P<0.01）.After targeting and inhibiting NRP1 gene in cells,the results showed that GATA3 protein expression was significantly higher after irradiation compared with NC.Compared with irradiation alone,knockdown of NRP1 gene while giving irradiation group cells showed no significant change in GATA3 protein expression.In summary,GATA3 acts as a positive regulator of the downstream target gene NRP1.The changes of GATA3 protein expression in radiation-induced fibrotic phenotype of lung epithelial cells were detected by using cytoplasmic protein separation and extraction assay,and the results showed that the expression of GATA3 protein in the nucleus of the cells was significantly elevated after irradiation,while the expression in the cytoplasm of the cells was significantly reduced.The cellular localization of GATA3 protein was detected by immunofluorescence,and the results showed that the entry of GATA3 protein into the nucleus increased significantly after irradiation.The JASPAR database was utilized to predict the specific binding site（AGATTG）of GATA3 on the NRP1 promoter,and the binding of transcription factor GATA3 to the NRP1 promoter was detected by Ch IP-qPCR.It showed that X-ray irradiation significantly enhanced the enrichment of GATA3 in the NRP1 promoter region,which mainly enriched in the NRP1 promoter region in A549 cells.It was mainly enriched in the NRP1 promoter region-127-252 bp（P<0.01）and in MLE-12 cells-262-402 bp（P<0.01）.The regulatory relationship of transcription factor GATA3 on NRP1 was detected by dual luciferase reporter gene assay,and the NRP1 promoter region was identified by specific binding site.p GL3-Basic vector was used to construct a recombinant plasmid for the NRP1 promoter region（a fragment of the first 700 bp）.The results showed that the luciferase activity in the GATA3OE+NRP1 promoter+p RL-TK group was significantly elevated（P<0.05）compared to the Vector+p GL3（+）+p RL-TK group.Taken together,this suggests that radiation upregulates the transcription factor GATA3,inducing it to enter the nucleus and bind to the NRP1 promoter region,which in turn regulates the expression of NRP1.To further elucidate the role of GATA3/NRP1 in RIPF,we detected the changes in the expression of EMT and fibrosis marker-related proteins and mRNAs after knocking down the GATA3 gene while overexpressing the NRP1 gene in the cells,and the results showed that compared with the NC group,the expression of epithelial markers E-cadherin protein and mRNA was significantly decreased in the IR group,and the mesenchymal markers N-cadherin and Vimentin,as well as fibrosis markersα-SMA and Collagen-1 protein and mRNA expression were significantly higher.Compared with IR,cells in the GATA3KD+IR group had elevated E-cadherin protein and mRNA expression and significantly lower N-cadherin,Vimentin,α-SMA and Collagen-1protein and mRNA expression.In contrast,E-cadherin expression was significantly elevated and N-cadherin,Vimentin,α-SMA and Collagen-1 protein and mRNA expression were significantly reduced in the GATA3KD-NRP1OE+IR group compared with the NRP1OE+IR group.This suggests that knockdown of GATA3 gene can effectively inhibit the developmental process of fibrosis,while overexpression of NRP1gene reversed this anti-fibrotic effect.When overexpressing the GATA3 gene and knocking down the NRP1 gene in cells for reverse validation,a opposite result to the above was obtained;overexpressing the GATA3 gene effectively promoted the developmental process of fibrosis,while knocking down the NRP1 gene reversed this pro-fibrotic effect.Taken together,this suggests that the transcription factor GATA3positively regulates NRP1 by binding to the NRP1 promoter region,thereby affecting the fibrosis process.5.To explorie the molecular mechanism of NRP1 gene regulation of radiation-induced pulmonary fibrosis（1）Firstly,the correlation between NRP1 and RIPF process was verified.Western Blot and q RT-PCR results showed that compared with the control group,the expression of NRP1 protein increased significantly with time after irradiation,and reached the highest at 24 weeks after irradiation,and the mRNA level increased to 5.62-fold（P<0.05）.IHC results showed that compared with the control group,brown NRP1-positive cells in lung tissue gradually increased with time after irradiation,and reached the highest at 24 weeks after irradiation.The IHC results showed that compared with the control group,brown NRP1-positive cells gradually increased in the lung tissue with the extension of irradiation,and reached the highest level at 24 weeks after irradiation,and the lung tissue was almost covered with positive cells.The changes of NRP1 protein and mRNA expression in radiation-induced fibrotic phenotype of lung epithelial cells were detected by Western Blot and q RT-PCR,and the results showed that the expression of NRP1 protein increased significantly after irradiation,and in A549 cells,NRP1 increased to 1.85-fold after irradiation（P<0.01）.In MLE-12 cells,NRP1 was elevated to 2.32-fold（P<0.01）.It suggests that the elevation of NRP1expression is related to the process of radiation-induced pulmonary fibrosis.（2）In vitro experiments confirmed the cytological mechanism of NRP1 gene regulation in radiological lung fibrosis.An immunoprecipitation method was used to detect the binding capacity of NRP1 and TGF-βin the radiation-induced fibrotic phenotype of lung epithelial cells,and the results showed that the binding capacity of NRP1 and TGF-βwas significantly enhanced after irradiation.The changes in the expression of EMT and fibrosis marker proteins and mRNAs after knocking down the NRP1 gene along with the addition of SRI-011381,an agonist of the TGF-β/Smad signaling pathway,were detected by using Western Blot and q RT-PCR.The results showed that the expression of E-cadherin in the irradiated group was significantly reduced,while N-cadherin,Vimentin,α-SMA and Collagen-1 expression was significantly elevated.Moreover,E-cadherin expression was elevated in the NRP1KD+IR group,N-cadherin,Vimentin,α-SMA and Collagen-1 protein expression was significantly reduced,E-cadherin expression in the NRP1KD+IR+SRI group was was significantly lower and N-cadherin,Vimentin,α-SMA and Collagen-1 protein expression was significantly higher.This suggests that knockdown of NRP1 gene can effectively inhibit the developmental process of fibrosis,while the addition of SRI-011381,an agonist of TGF-β/Smad signaling pathway,reversed this anti-fibrotic effect.Taken together,this suggests that NRP1 regulates the process of fibrosis through the TGF-β/Smad signaling pathway.6.To explore the mechanism of m6A modification during the formation of radiation-induced fibrotic phenotypes in lung endothelial cells and fibroblasts（1）First,human endothelial cell HVUEC and human fibroblast HFL-1 cells were irradiated with single X-ray to construct radiation-induced fibrotic phenotypes of lung endothelial cells and fibroblasts.The results showed that the total m6A level was significantly increased in the radiation-induced lung endothelial cell fibrosis phenotype,while the total m6A level was decreased in the radiation-induced lung fibroblast fibrosis phenotype.（2）The protein and mRNA expression changes of m6A modifying enzyme VIRMA,transcription factor GATA3,and downstream target gene NRP1 in radiation-induced fibrotic phenotypes of lung endothelial cells and fibroblasts were detected using Western Blot and q RT-PCR.The results showed that in radiation-... |
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