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

Highlights

• MEN1 gene was negatively correlated with lung fibrinogen markers.

• MEN1 gene regulates radiation induced EMT and ECM processes.

• Menin protein interacts with Smad2 inhibition the progression of radiation pulmonary fibrosis.

• MEN1 can enhance the effect of radiation induced pulmonary fibrosis.

Abstract

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 (α-SMA), Collagen-1 and transforming growth factor (TGF-β) 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 α-SMA, Collagen-1, and TGF-β/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-β up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of α-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extracellular 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-β signaling pathway, and down-regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radionuclide fibrosis.

Conclusion

MEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-β and the activation of TGF-β/Smads signaling pathway.

Introduction

Radiotherapy has been a common method for clinical treatment of malignant tumors. However, for lung tumors, radiation therapy may cause radiation pneumonitis, which further causes radiation pulmonary fibrosis. Radiation pulmonary fibrosis is one of the most difficult complications of radiation therapy. How to reduce the occurrence of radiation pulmonary fibrosis and the degree of pulmonary fibrosis to enhance the effect of radiotherapy has become a hot spot for cancer radiotherapy. Studies have shown that radiation-induced pulmonary fibrosis is the result of the combined effects of multiple effector cells, cytokines, and inflammatory mediators (Kamp, 2003; Pardo & Selman, 2016), of which transforming growth factor (TGF-β) is considered to be one of the most important mediators (Castellone & Laukkanen, 2017).

MEN1 gene, discovered by Chandrasekharappa et al. (1997), is located on chromosome 11q13 (Larsson et al., 1988; Thakker et al., 1989). The MEN1 gene is 9 kb in length and contains 10 exons encoding 610 amino acids (Bertolino et al., 2003a). The MEN1 gene encodes protein menin, which has a wide distribution but differs in its expression. Studies have shown that the menin protein may act as a signaling molecule to regulate gene expression. Since it can binds directly to double-stranded DNA, the menin protein has a variety of biological functions, such as affecting cell proliferation, cell migration, DNA damage repair, and apoptosis. Moreover, in different species of organisms, the protein has a higher homology (Karges et al., 1999; Khodaei et al., 1999; Guru et al., 2001).

According to the current literatures, menin recruits Smad3 and regulates the expression of Smad3 target genes. Menin can bind directly onto Smad3, a downstream response element of the TGF-β pathway (as demonstrated in COS cells, COS cells are kidney fibroblasts), to modulate TGF-β signaling pathway and the process of TGF-β induced genetic transcription (Kaji et al., 2001). The knockdown of menin decreases the inhibitory effect of TGF-β-induced proliferation (Hussein et al., 2008). Knockdown of Menin can also reduce the binding of Smad3 to DNA (as evidenced by the secretory tumor cell line in the pituitary). On the other hand, it has been reported that TGF-β can upregulate the expression of menin in MLL-AF9 transformed mouse bone marrow cells. When the TGF-β receptor TGF-βRII was knocked out conditionally in mice, the expression of menin was down-regulated in the liver samples (Zhang et al., 2011; Feng et al., 2017a).

However, whether the MEN1 gene can regulate pulmonary fibrosis through TGF-β signaling pathways is still unclear. Activation of fibroblasts, accumulation of extracellular matrix of myofibroblasts and transformation of epithelial mesenchymal cells during pulmonary fibrosis are important mechanisms of pulmonary fibrosis. What role MEN1 plays in these critical steps and whether it regulates the processes of epithelial mesenchymal transition and extracellular matrix aggregation are also unknown. Here, we used 20 Gy X-rays to irradiate mouse lungs to form a mouse model of pulmonary fibrosis. The results showed that the MEN1 gene was negatively correlated with lung fibrinogen markers. Using mouse embryonic fibroblasts and mouse alveolar epithelial cell lines demonstrated that the MEN1 gene regulates EMT and ECM processes, and that the menin protein interacts with Smad2, a downstream response element of TGF-β, there by Inhibition the progression of radiation pulmonary fibrosis.