RESUMO
Radiotherapy is widely used in the management of lung cancer, and physicians are aware that the effect of radiotherapy is dependent on radiosensitivity. Although a series of blockers and activators targeting molecules related to radioresistance have been developed as radiation sensitizers, compensatory mechanisms or drug resistance limits their clinical efficacy. The identification of a key molecule related to lung cancer cell radioresistance or an effective molecular target is a challenging but important problem in radiation oncology. A previous study found that neuropilin 1 (NRP1) is related to radioresistance in A549 cells and is associated with VEGF, PI3K-Akt, MAPK-ERK, P38, NF-κß and TGF-ß. Inhibition of NRP1 can increase the radiosensitivity of A549 cells. Therefore, NRP1 may be a molecular target for radiotherapy-sensitizing drugs in lung cancer. The present study investigated the key downstream genes of NRP1, verified their regulation and clarified their roles in regulating lung cancer radioresistance. NRP1 positively regulated the downstream homeobox genes (HOXs) HOXA6, HOXA9 and mixed lineage leukaemia 5 (MLL5) in addition to MLL5-regulated HOXA6 and HOXA9, but these genes did not regulate NRP1. MLL5, HOXA6 and HOXA9 levels were decreased in tumour tissues and positively correlated with NRP1. All of these genes were induced by ionizing radiation in vivo and in vitro. NRP1 expression was significantly lower in squamous cell carcinoma compared with that in adenocarcinoma, and lymph node metastasis occurred more often in patients with lung cancer with high MLL5 and NRP1 expression compared with patients with low MLL5 and NRP1 expression. Collectively, these data confirmed that NRP1 is associated with MLL5 and regulates radioresistance through HOXA6 and HOXA9.
RESUMO
Background: Neuropilin 1 (NRP1) is a pleiotropic receptor which can interact with multiple ligands and their receptors. It plays an important role in the process of axonal growth, angiogenesis, tumor metastasis and radiation resistance in endothelial cells and some tumor cells. Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis, and has received considerable attention in recent years. Material and Methods: In this study, A549 lung cancer cell lines with different NRP1 expression levels were constructed in vitro, a two-dimensional (2D), three-dimensional (3D) co-culture system and tumor-bearing model was established in SCID mice. Western blot, qRT-PCR, immunofluorescence, cytometric bead array and flow cytometry were used to investigate the effect of the tumor microenvironment in NRP1-induced lung cancer cell radiation resistance. Results: In 2D or 3D co-culture system, NRP1 could be regulated inflammatory factors such as TNF, IL-6 IL-8 and IL-17 and the related chemokines MCP-1, IP-10 and RANTES in the tumor microenvironment, which in turn induced radiation resistance in lung cancer cells. In addition, different expression levels of NRP1 in 2D, 3D culture systems and tumor-bearing models were able to significantly regulate cell phenotype, proliferative capacity, epithelial-mesenchymal transition (EMT) and the radiation resistance of A549 cells. Conclusion: Our results verified that NRP1, inflammatory factors, chemokines and related signaling pathways, which affect the transformation of related cell components and thus lung cancer cell immune tolerance and migratory ability, all play an important role in radiation resistance.
RESUMO
Exposure to ionizing radiation often induces T helper (Th) cell differentiation, resulting in an imbalance of Th1 and Th2 cellular subtypes, which can affect the efficacy of cancer radiotherapy. The aim of this study was to analyze differential expression of Th1, Th2 and Th3/Type 1 regulatory T cell (Tr1) subtype-related genes and cytokines in mouse thymocytes after high- and low-dose systemic radiation, using functional classification gene arrays and Elisa assays, and to explore the molecular mechanisms underlying radiation's immune effects and their relationship with Th1/Th2 immunity. We found that expression of 8 genes was upregulated after LDR, while expression of 5 genes was downregulated. After HDR, 54 genes were upregulated and 3 genes were downregulated, including genes related to Th1, Th2 and Th3/Tr1 cellular subtypes, Th1/Th2-type immune response genes and transcription factor-related genes. In the foregoing results, LDR and HDR in the thymus induced opposite patterns of expression for Th1-, Th2- and Th3-type related cytokines TGF-ß, C/EBP-ß and TNF-α. We also found that expression of Interferon-γ (IFN-γ) and Interleukin-2 (IL-2), which have a moderating effect on immune function, was upregulated after LDR. Furthermore, the secretion of negative regulatory factors Interleukin-1ß (IL-1ß), Interleukin-4 (IL-4), transforming growth factor-ß (TGF-ß) and Interleukin-21 (IL-21) was reduced after LDR, but HDR produced the opposite effect and stimulated their expression. These findings suggest that LDR may induce a Th1-type immune response, while HDR may lead to a Th2-type immune response.
Assuntos
Diferenciação Celular , Citocinas/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Animais , Diferenciação Celular/efeitos da radiação , Citocinas/genética , Relação Dose-Resposta à Radiação , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Masculino , Camundongos Endogâmicos ICR , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos da radiação , Timócitos/metabolismo , Timócitos/efeitos da radiação , Timo/metabolismo , Timo/efeitos da radiação , Fatores de Transcrição/metabolismoRESUMO
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 8â¯weeks and weighing 18-22â¯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 20â¯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 24â¯weeks after irradiation. The expression of MEN1, smooth muscle actin (α-SMA), Collagen-1 and transforming growth factor (TGF-ß) 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 10â¯Gy X-rays. The expression of α-SMA, Collagen-1, and TGF-ß/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 4-8â¯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-ß 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-ß 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-ß and the activation of TGF-ß/Smads signaling pathway.