| Lung cancer is one of the most common malignant tumors in the world.In recent years,more and more focuses of malignant tumors have shifted from the tumor cells themselves to the tumor microenvironments during the development process.Radiation therapy and chemical therapy is currently a common treatment of cancer.In radiotherapy,the sensitivity of tumor cells to ionizing radiation killing determines the efficacy of the final radiotherapy,which is mainly due to the radiation resistance of the tumor cells itself,while radiotherapy is also affected by the tumor microenvironment.Therefore,to deepen the understanding of the relationship between radiation resistance and tumor microenvironment of tumor cells,and intervene with a targeted therapy on tumor cells with radiation resistance or tumor stem cells,to achieve the purpose of effective treatment of cancer,is the focus and difficulty in the treatment of tumor.Research background:Neuroplin 1,NRP1,is an important member of the family of neurofilmins and often appears on the surface of endothelial cells or tumor cells and can play an important role in promoting angiogenesis and tumor metastasis.In recent years,a number of studies have shown that NRP1 in lung cancer,breast cancer,liver cancer,pancreatic cancer and other tumor tissue is widely expressed,and closely related to poor prognosis [1].It was found that the expression of NRP1 was related to the radiation resistance of A549 cells and could exert its radiation resistance by activating signal pathway VEGF,PI3K-Akt,MARK-ERK,NF-?B and TGF-?.Therefore,NRP1 may play an important role as an effective molecular target in future lung cancer radiotherapy,radiation sensitization drug screening and so on.Tumor microenvironment is the internal environment of tumor cells in the process of tumor development.It is composed of tumor cells,stromal cells,infiltrating cells and related cytokines.With the interaction between tumor cells and its surrounding microenvironment,tumor proliferation,adhesion,angiogenesis and invasion are accelerated.So are the occurrence and development of tumors.Therefore,in-depth study of tumor microenvironment can provide new ideas for tumor radiotherapy.Three-dimensional culture in recent years has been widely used in biology and drug research,etc.,compared to the previous two-dimensional culture system,cell morphology and biological function from it are closer to the body's living cell status.At present,three-dimensional culture system is mainly used in the study of molecular mechanism,angiogenesis,tumor microenvironment and tumor stem cells in tumor oncology.Research objectives: 1.Three-dimensional model of cell culture was constructed and to verify the morphology and the expression of related proteins;.2.To investigate the role of tumor inflammatory microenvironment in NRP1-induced radiation resistance and its molecular mechanism;3.To investigate the role of tumor microenvironment in NRP1-induced radiation resistance and the mechanism of lung cancer cell metastasis.Research methods: 1.The NRP1Low-A549 and A549-RR(radiation-resistant)cell models has been constructed.The expression of NRP1 at the transcription level and protein was verify by real-time PCR and Western blot.the three-dimensional culture model of A549 cells was constructed by Matrigel matrix gel,and detected the change of cell morphology in three-dimensional culture model and the related protein expression by immunofluorescence;2.The construction of the different cell lines and Jurkat cells(human T lymphocytes) three-dimensional co-culture model,10 Gy X-ray and the supernatant was collected after 48 h,using the CBA method to detect the expression changes of related inflammatory cytokines by flow cytometry,investigate the tumor inflammatory microenvironment in NRP1-induced radiation resistance of lung cancer cells;3.The co-culture model of human peripheral blood lymphocytes and A549 cells was established.After 6 Gy X-ray irradiation,the differentiation of regulatory T cells in tumor inflammatory microenvironment was detected by flow cytometry and ELISA.Secretion and the expression of IL-10,TGF-? and IL-17,and to elucidate the immune tolerance mechanism of tumor inflammatory microenvironment in radiation resistance;4.Construction of A549,NRP1Low-A549,A549-RR cells and HLF-1 cells(human embryonic lung fibroblasts)three-dimensional co-culture model,The supernatant was collected after 10 Gy X-ray for 48 h,and collected by CBA method.To investigate the effect of tumor microenvironment on NRP1-induced the radiation resistance of lung cancer cells in vitro;5.The effect of NRP1 on the expression of ionizing radiation-related factors(TGF-?)in tumor microenvironment was detected by ELISA.The expression of CAFs(tumor-associated fibroblasts)and ?-SMA were detected at the transcriptional level and protein level respectively.Expression of TGF-? pathway-related factors.Finally,Transwell method was used to detect the effect of NRP1 on the migration ability of A549 cells in tumor microenvironment.Research results: 1.Successful establishment of three-dimensional cultured cell model The expression of NRP1 in NRP1Low-A549 and A549-RR cell models was verified by Real-time PCR method and Western blot method.It was found that the level of NRP1 in A549-RR cell line was significantly higher than A549 cells wild type,while NRP1Low-A549 cell line the NRP1 in the transcription level and protein were significantly lower than A549 cells wild type.On the basis of this,the three-dimensional culture model of A549 cells was successfully constructed by Matrigel matrix gel.The morphology of the cells was three-dimensional tumor under the optical microscope.The internal cells were round and regular,and some cells migrated from the tumor spheres.The morphological changes of three-dimensional cultured cells were detected by immunofluorescence.The results showed that the cytoskeleton was contracted and denser,and the cells were widely connected and the cell morphology changed obviously.At the same time,the expression of EMT-related markers(Vimentin,N-cadherin and E-cadherin)was significantly changed.On the basis of this,A549,NRP1Low-A549 and A549-RR were co-cultured with Jurkat cell three-dimensional co-culture model(tumor inflammatory microenvironment)and three-dimensional co-culture model of HLF-1 cells(tumor migration microenvironment).2.The role of tumor inflammatory microenvironment in NRP1-induced radiation resistance of lung cancer cells The expression of IL-12p70 and TNF in A549-RR group was decreased(p<0.05),and in NRP1Low-A549 group was increased(p<0.05),and the expression in A549-RR group was significantly higher than that in NRP1Low-A549 group(p<0.05).The expression of IL-6 and IL-8 in A549-RR group was significantly higher than that in group A549(p< 0.01),while the expression of the two factor was reversed in NRP1Low-A549 group(p<0.05).And after ionizing radiation,the expression changes of the two groups before and after irradiation were the same as those in the A549-RR group(p<0.05),which was significantly increased in the NRP1Low-A549 group(p<0.05).The expression of IL-1 and IL-10 did not change much in the two groups,but decreased after irradiation(p<0.05).Suggest that inflammatory cytokines NRP1 in tumor inflammatory microenvironment after radiation increased positively regulated(IL-12p70 and TNF),and reduced negative inflammatory cytokine regulation(IL-6 and IL-8),so as to improve the radiation resistance of lung cancer cells.3.Effects of ionizing radiation on Treg cells and related factors in tumor microenvironment To investigate the effect of ionizing radiation on the immune tolerance in the inflammatory microenvironment of the tumor,we used flow cytometry and ELISA to differentiate,co-stimulate the expression of Treg cells and the secretion of related cytokines in tumor inflammatory microenvironment To detect.The results showed that the expression of CD4+CD25+,CD4+CD25+Foxp3+ and CD4+CD25+NRP1+ in the inflammatory microenvironment of the irradiated tumor increased(p<0.05),which indicated that the differentiation of Treg cells was promoted.The expression of ICOS and CTLA-4 was up-regulated(p<0.05),but only IL-17 secretion was significantly higher in the cytokines(p<0.05),while IL-10 and TGF-? were not up-regulated.The These results suggest that NRP1 induces the radiation resistance of lung cancer cells through the immune tolerance mechanism of regulatory T cells in tumor inflammatory microenvironment.4.The mechanism of tumor microenvironment in NRP1-induced radiation resistance of lung cancer cells The expression of RANTES and MCP-1 in A549-RR group was significantly lower than that in A549 group(p<0.05),and increased in the NRP1Low-A549 group(p<0.05)After ionizing radiation,the two factors in each group compared with the control group were reduced to varying degrees(p<0.05).In contrast,the expression levels of IP-10 and CXCL8(IL-8)in the other groups were lower than those in the A549 group,and decreased significantly in the NRP1Low-A549 group(p<0.05).(p<0.05),and the expression of CXCL8 was decreased in the A549-RR group(p<0.05),and in the NRP1Low-A549 group was increased(p<0.05)The expression of IP-10 was decreased in both groups compared with that before irradiation,and the difference was statistically significant(p<0.05).The results showed that NRP1 induced radiation resistance may be involved in the regulation of tumor chemotaxis in tumor microenvironment,such as RANTES,MCP-1,IP-10 and CXCL8 factors,and then activate the relevant signaling pathway to enhance the migration of lung cancer cells ability to ultimately inhibit the radiation sensitivity of lung cancer cells.5.Effects of tumor microenvironment on migration ability and intercellular interaction of A549 cells In order to investigate the mechanism of NRP1 on tumor cell migration in tumor microenvironment,the expression of TGF-? in NRP1-regulated tumor microenvironment was detected by ELISA.NRP1 could inhibit the expression of TGF-? in A549 cells(TGF-?,Smad7)in fibroblasts,and then transformed fibroblasts into CAFs cells.The migration of A549 cells was detected by Transwell method.The results showed that the migration ability of A549 cells was significantly affected by the co-culture(P <0.05),and the migration ability of A549 cells was significantly increased with the increase of NRP1 The It can be seen that NRP1 enhances the migration of TGF-? signaling pathway by enhancing the synthesis and secretion of chemokines,inducing the transformation of CAFs and enhancing the migration ability of lung cancer cells in tumor microenvironment.Conclusions: 1.The three-dimensional cell culture model successfully established by Matrigel matrix gel can simulate the microenvironment and biological characteristics of tumor cells in vivo compared with the traditional two-dimensional culture.2.In the inflammatory microenvironment of the tumor,ionizing radiation up-regulates positive regulatory inflammatory cytokines(IL-12p70 and TNF)and down-regulates the negative regulatory inflammatory cytokines(IL-6 and IL-8)NRP1-induced radiation resistance of lung cancer cells;3.Radiation can induce Treg cell differentiation in tumor inflammatory microenvironment and regulate the immune tolerance of tumor by enhancing the expression of its costimulatory molecule and the secretion of IL-17.4.In the tumor microenvironment,NRP1 enhances the radiation resistance of lung cancer cells through the regulation of chemokines such as RANTES,MCP-1,CXCL8 and IP-10.4.In the tumor microenvironment,NRP1 can enhance the migration ability of lung cancer cells by regulating the secretion of TGF-? signaling pathway and induce the transformation of CAFs,and further influence NRP1 on the radiation resistance of lung cancer cells. |
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