The Mechanism Of FOXO1/MEN1 Pathway In Radiation-induced Pulmonary Fibrosis

Radioactive-induced pulmonary fibrosis(RIPF)is a common complication of radiotherapy for thoracic tumors.It is also one of the important reasons that limit the therapeutic effect of radiotherapy on thoracic tumors.The main pathological manifestations of RIPF are inflammation caused by early acute radiation injury,including enlargement of lung texture,enlargement of alveolar septum and inflammatory infiltration,and fibrosis symptoms in late stage,including increase of fibrous connective tissue,destruction of alveolar structure and decrease of parenchymal cells.Because of the unpredictability and irreversibility of radiation-induced pulmonary fibrosis,it is difficult to treat it and there is a lack of effective therapeutic drugs in clinic.Background:MEN1 gene is located on chromosome 11q13,and its mutation can cause multiple endocrine neoplasm type 1(MEN1)syndrome.MEN1 syndrome is a dominant hereditary disease characterized by tumorigenesis of endocrine organs,including pituitary,parathyroid and islet.The MEN1 gene encodes only one protein,called menin protein.Menin protein is very important in the development of various tissues and organs.Complete deletion of MEN1 gene can cause mouse embryo death.Studies have shown that TGF-beta is one of the most widely studied fibroblast-promoting cytokines.Menin regulates TGF-beta signal transduction by interacting with Smad3,a downstream effector of TGF-beta,and inhibits the binding of Smad3/4 at specific transcriptional regulatory sites to regulate its expression.At present,the study of MEN1 gene is mostly focused on the occurrence and development of tumors,and there are few reports about MEN1 and radiation pulmonary fibrosis.Purposes:The treatment of radiation pulmonary fibrosis has become one of the hotspots in the field of radiobiology.To elucidate the mechanism of radiation pulmonary fibrosis is of great significance to prevent and treat radiation pulmonary fibrosis,and even to improve the curative effect of radiotherapy for thoracic malignant tumors.Methods:Based on the above research status and experimental results,this study first established a mouse model of radiation-induced pulmonary fibrosis.The success of RIPF model was verified by H&E and Mason staining.Western blot,q RT-PCR and immunohistochemistry(IHC)were used to detect whether MEN1 gene participated in RIPF process.In vitro experiments,Mice-derived embryos were knocked out by using MEN1 gene.At the same time,si RNA-MEN1 specifically inhibited the expression of MEN1 gene in mouse lung epithelial cell line(MLE-12).Western blot,q RT-PCR and IF techniques were used to further detect the effect of MEN1 on RIPF markers.Finally,the important signal pathways of RIPF were verified by IP and CHIP techniques.To clarify the molecular mechanism of its regulation.Results:1.Confirmation of the role of MEN1 in the formation of radiation-induced pulmonary fibrosis(1)In this study,a single 20 Gy X-ray was used to irradiate the thorax of mice to establish a model of pulmonary fibrosis in mice.The degree of lung injury in mice was detected by H&E staining.The results showed that the alveolar structure of the control group was intact and the alveolar wall was fine.In RIPF model group,acute radiation injury occurred 4-8 weeks after irradiation,alveolar septum widened and pulmonary texture thickened,alveolar fusion,alveolar integrity decreased,interstitial cells increased and inflammatory cells infiltrated 16 weeks after irradiation.At 24 weeks after irradiation,obvious fibrous tissue proliferation,massive lung consolidation and finally pulmonary fibrosis were observed.(2)Masson staining was used to validate the RIPF model.The results showed that the alveolar structure of the control group was clear,there was no blue collagen deposition and no obvious fibrosis.Collagen deposition(blue)began to appear in the lungs of mice in model group around 8 weeks after irradiation,but there was obvious local fibrosis area around bronchioles at 16 and 24 weeks after irradiation.Large amount of blue collagen deposition was observed,suggesting that the model of radiation-induced pulmonary fibrosis in mice was successfully established.(3)Western blot,q RT-PCR and IHC were used to detect the expression of MEN1 and fibrosis markers in lung tissue of RIPF model.The results showed that the expression of MEN1 decreased significantly with the passage of time after irradiation,reaching the lowest level 24 weeks after irradiation,while the expression of pulmonary fibrosis markers alpha-SMA and Collagen-1 increased gradually with the prolongation of time after irradiation.These results suggest that the decrease of expression of MEN1 is related to radiation-induced pulmonary fibrosis in mice,and MEN1 may play an important role in the formation of radiation-induced pulmonary fibrosis.2.In vitro study of the effect of MEN1 gene on radiation-induced pulmonary fibrosis(1)In vitro experiments confirmed the cytological mechanism of MEN1 gene regulating radiation-induced pulmonary fibrosis.The expression of MEN1 in lung epithelial cells(MLE-12)was inhibited to detect the changes of pulmonary fibrosis markers.The results showed that the expression of E-cadherin,a marker of epithelial cells,was significantly decreased and the expression of vimentin,a marker of mesenchymal cells,was enhanced after targeted inhibition of MEN1.The inhibition of MEN1 gene could further promote the expression of vimentin after irradiation,indicating that the inhibition of MEN1 gene could enhance the process of radiation-induced epithelial-mesenchymal transition(EMT)and accelerate it.Dermal cells transformed into mesenchymal cells.(2)To elucidate the role of MEN1 gene in the transformation of fibroblasts into myofibroblasts,embryonic fibroblasts knocked out of the MEN1 gene(MEF-MEN1 Delta/Delta)were used.Deletion of MEN1 gene can significantly promote the expression of alpha-Smooth Muscle Actin(ACTA2)in MEF cells,which indicates that deletion of MEN1 gene can promote the differentiation of fibroblasts into myofibroblasts,and the expression of Collagen-1,the main component of ECM in myofibroblasts,is also significantly enhanced.After irradiation,it was found that the knockout of MEN1 gene could significantly enhance the irradiation effect and promote pulmonary fibrosis.(3)The stable overexpression of MEN1 gene in MEF cell model(MEF-MEN1 high)was further constructed to study whether MEN1 gene can regulate the differentiation of fibroblasts into myofibroblasts.The results showed that the overexpression of MEN1 gene could significantly inhibit the expression of alpha-SMA and the activation of fibroblasts,and the expression of N-cadherin and Vimentin in mesenchymal cells decreased significantly,while the expression of E-cadherin in epithelial cells increased.IF results showed that the fluorescence intensity of a-SMA increased significantly after irradiation,indicating that fibroblasts differentiated into myofibroblasts after irradiation,but this differentiation was significantly inhibited in MEF cells with over-expression of MEN1 gene,indicating that the over-expression of MEN1 gene could inhibit the process of radiation-induced pulmonary fibrosis and play an important role in the process of fibroblast differentiation into myofibroblasts.Use.Further studies showed that the fluorescence intensity of Collagen-1 was significantly enhanced after irradiation,but the fluorescence intensity of Collagen-1 was significantly inhibited after the overexpression of MEN1 gene.These results suggest that the overexpression of MEN1 may inhibit the process of RIPF by inhibiting the accumulation of Collagen-1 in ECM.3.Molecular mechanism of FOXO1/MEN1 regulating radiation-induced pulmonary fibrosisWhat signaling pathway does MEN1 gene regulate the progression of pulmonary fibrosis? In this study,the molecular mechanism of radiation-induced pulmonary fibrosis signaling pathway was explored.(1)Smad2,Smad3 and Smad7,the key downstream signal components of TGF-beta signaling pathway,were detected in this study.The results showed that the deletion of MEN1 gene could significantly promote the expression of Smad2/3,inhibit the expression of Smad7,an inhibitor of TGF-beta signaling pathway,and then promote the signal transduction of TGF-beta.IP experiments showed that menin protein interacted with Smad2 protein.However,MEF-MEN1 high assay showed that the overexpression of MEN1 gene inhibited the expression of Smad2/3 protein,and played an important role in TGF-beta/Smads signal transduction.The decrease of Smad2 expression can inhibit the activity of TGF-beta signaling pathway and achieve the goal of inhibiting pulmonary fibrosis.(2)To verify whether FOXO1,a transcription factor upstream of MEN1,regulates the progression of radiation-induced pulmonary fibrosis by regulating the expression of MEN1 gene? Firstly,the expression of FOXO1 in the lung tissue of RIPF model mice was detected.The results showed that the expression of FOXO1 in the lung tissue of RIPF model mice gradually decreased with time and reached the lowest level after 24 weeks of irradiation.Combining with the previous experimental results,the expression trend of FOXO1 was contrary to that of pulmonary fibrosis markers,but consistent with that of MEN1 gene.After targeted inhibition of FOXO1 in MEF cells,we found that the protein and m RNA levels of MEN1 gene decreased significantly.To verify the regulatory mechanism of post-irradiation transcription factor FOXO1 on MEN1 gene,the binding of FOXO1 protein to the promoter region of MEN1 gene was detected by Ch IP technique.The results showed that the binding of FOXO1 protein to the promoter region of MEN1 gene was significantly reduced after irradiation compared with the control group.These results suggest that the binding of FOXO1 to MEN1 gene is significantly inhibited after ionizing radiation,which results in the decrease of the expression of MEN1 gene and promotes the occurrence and development of pulmonary fibrosis.Conclusion:1.MEN1 plays a important role in the process of radiation-induced pulmonary fibrosis in mice,and its expression decreases gradually with the prolongation of irradiation time.2.Targeted inhibition of MEN1 gene promotes the process of epithelial mesenchymal transformation(EMT)and radiation-induced pulmonary fibrosis.3.The knockout of MEN1 gene can promote the differentiation of fibroblasts into myofibroblast cells,promote the accumulation of ECM and promote radiation pulmonary fibrosis.4.Overexpression of MEN1 gene can inhibit the expression of important markers of pulmonary fibrosis and alleviate radiation-induced pulmonary fibrosis.6.Menin can interact with Smad2,inhibit the expression of Smad2 protein,inhibit the signal transduction of TGF-beta/Smads,and inhibit radiation pulmonary fibrosis.7.Targeted inhibition of FOXO1 can reduce the expression of MEN1.In addition,ionizing radiation can inhibit the binding of FOXO1 protein to the promoter region of MEN1 gene,inhibit the expression of MEN1,and promote the development of radiation-induced pulmonary fibrosis.