Mechanism Of Radiation-induced Exosomal MiR-4655-5p Mediating Macrophage Proliferation And Inflammatory Response

Objectives:When patients with non-small cell lung cancer receive radiotherapy,radiation acts on tumor cells and alveolar epithelial cells within the radiation volume.In addition to directly causing the death of cancer cells,studies believe that radiotherapy can also recruit macrophages to reach the site of injury by releasing signal molecules,and release inflammatory cytokines,thus affecting the therapeutic effect and patient survival.However,the specific communication mechanism between macrophages and lung cancer cells is not fully understood.Exosome miRNA is one of the important ways to mediate intercellular communication.Differential activation of macrophages is regulated by miRNA.Therefore,this study was designed to explore whether exosomes secreted by normal lung epithelial cells and lung cancer cells after irradiation can activate macrophages.Furthermore,the signaling mechanism of irradiated tumor-derived exosomes promoting macrophage proliferation and regulating inflammatory response after irradiation was further studied.Methods:We evaluated the degree of infiltration of different types of immune cells in lung tumor tissue after radiotherapy,lung tumor tissue without radiotherapy and normal lung tissue by bioinformatics methods,and analyzed the relationship between immune cell infiltration and prognosis of tumor patients.The rat model of radiation pneumonitis was constructed,and the lung inflammation and macrophage infiltration were observed by HE staining,immunofluorescence staining.THP-1 cells were induced to differentiate into macrophages using phorbol-12-myristic acid-13-acetic acid(PMA).The optimal concentration and duration of PMA were determined by cell viability test and attachment observation.Macrophages were identified by microscopic observation of cell morphology,detection of CD11 b expression by q RT-PCR and detection of cell surface marker CD68 by flow cytometry.The exosome was isolated by differential centrifugation.The morphological characteristics of the exosome were observed by transmission electron microscopy,the expression of exosome marker proteins was detected by western blot,and the particle size and distribution of the exosome were analyzed by a nanoparticle tracking analyzer.The exosomes were co-cultured with macrophages,and the proliferation of macrophages was detected by CCK-8 kit.The m RNA expression levels of cell inflammation-related factors were detected by q RT-PCR.The protein chip was used to characterize the changes in the expression profile of inflammatory factors,and ELISA kit was used to verify the changes in inflammatory factors.High-throughput miRNA chip analysis was used to screen miRNA with up-regulated expression and q RT-PCR verification was conducted.The miRNA functions were verified by transfecting miRNA mimics/inhibitors to conduct function acquisition and function deletion simulation experiments.Bioinformatics analysis was used to predict the target genes of miRNA,and the double luciferase reporter gene experiment was conducted to verify the targeting relationship.Further,RT-q PCR and western blot were used to verify the expression of the relevant pathway regulatory proteins of the target genes.sh RNA was used to knock down the target gene and verify the function of the target gene.Results:Data mining based on correspondence analysis revealed that in the clinical practice of radiotherapy,M0 macrophages in the lung of patients after radiotherapy were increased,and showed a moderate negative correlation with dendritic cells.Meanwhile,high infiltration of macrophages and neutrophils was positively associated with better patient survival.The in vivo experiments in rats showed that ionizing radiation in the lungs would lead to the increase of macrophages and activate the M1 polarization of macrophages.We studied the function of exosome-mediated macrophages in vitro.First,the macrophages were successfully induced and identified,and the exosomes derived from the cell supernatant were isolated and identified.Under the co-culture system of exosome and macrophage,the exosome secreted by normal lung epithelial cells and lung cancer cells can promote macrophage proliferation and secretion of inflammatory factors.Besides,the exosomes secreted by the above two cells played a more significant role in promoting the biological behavior of macrophages after radiation exposure.We found and confirmed that exosomes secreted by lung cancer cells after radiation can transport miR-4655-5p into macrophages and induce and activate macrophage-related functions through miR-4655-5p.Further verification experiments of miR-4655-5p overexpression/inhibition confirmed that miR-4655-5p could promote macrophage proliferation and regulate the secretion of inflammatory factors.Therefore,miR-4655-5p has a direct targeting relationship with MID1/PP2 Ac.Conclusions:This study illustrates that normal lung epithelial cells and lung cancer cells are exposed to exosomes as mediators to activate macrophages and promote their proliferation and secretion of inflammatory factors through the ionizing radiation induced bystander-effect.miR-4655-5p in exosomes secreted by lung cancer cells after irradiation is a signaling molecule that promotes the proliferation of macrophages and regulates their inflammatory response,and the direct targeting relationship between miR-4655-5p and MID1/PP2 Ac has been confirmed.A communication mechanism between lung cancer cells and macrophages under ionizing radiation was revealed.