Mechanism of MEN1 gene in radiation-induced pulmonary fibrosis in mice

Objective: To investigate the regulatory mechanism of MEN1 gene in radiation-induced lung fibrosis in mice and provide a new theoretical basis for the clinical treatment of radiation pulmonary fibrosis. Methods: First, 80 C57BL/6 mice aged 8 weeks and weighing 18-22 g were selected, half of them were male and the other half were female. The mice were divided into control group and irradiation group (40 mice in each group) according to the method of the random number table. A radiation-induced lung fibrosis mouse model was established in which a single X-ray irradiation of 20 Gy was applied to the right lung in the irradiation group; H& E and Masson staining were used to verify whether the model was successful at 4, 8, 16 and 24 weeks after irradiation. The expression of MEN1, smooth muscle actin (alpha-SMA), Collagen-1 and transforming growth factor (TGF-beta) in lung tissue were detected by Western blot and qPCR. Secondly, in the mouse embryonic fibroblast cell line (MEF) and mouse lung epithelial cell line (MLE-12), we constructed cell models of MEN1 knockout and interference separately with the irradiation of 10 Gy X-rays. The expression of alpha-SMA, Collagen-1, and TGF-beta/Smads signaling pathway molecules was detected by qPCR. Finally, using the immunoprecipitation (IP) method, we can detect the interaction between Smad2 and the protein menin encoded by the MEN1 gene. Results: The results of the radiation pulmonary fibrosis model in mice showed that compared with the control group, the alveolar septum widens, the alveolar integrity decreases, the lung tissue slightly thickens, and a small amount of collagen deposits appear after 4-8 weeks in the model group. At twenty-fourth weeks, a large number of cells in the interstitial space of the lung tissue and a localized focal fibrosis area were observed. Further study found that radiation induced fibrogenic inflammatory cytokines TGF-beta up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of alpha-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extra cellular matrix was overconcentrated and eventually promoted the formation of pulmonary fibrosis. In vitro, we found that knockout and interference of MEN1 gene can significantly enhance radiation-induced fibrosis, and up-regulate the expression of downstream molecules Smad2 and Smad3 of TGF-beta signaling pathway, and down regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radio-nuclide fibrosis. Conclusion: MEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-beta and the activation of TGF-beta/Smads signaling pathway.