Mechanism Of NRP1 On Radiation Induced EMT In Lung Cancer Cells

Lung cancer is one of the most malignant tumors with the fastest growth rate of morbidity and mortality in human cancers,and it is currently the leading cause of cancer death in humans.The study found that neuropilin-1?NRP1?is closely related to the occurrence and development of tumors.Epithelial-mesenchymal transition?EMT?also plays an important role in embryonic development,wound healing,tissue regeneration,organ fibrosis,and cancer progression.One of the main side effects in radiation therapy for patients with IR-induced pulmonary fibrosis,EMT plays an important role in the progression of IR-induced pulmonary fibrosis.Therefore,to explore the role of NRP1 in IR-induced lung cancer cell EMT and its molecular mechanism,it has important guiding significance for reducing the side effects of radiotherapy for lung cancer.Background:NRP1 is a transmembrane glycoprotein located on axons of nerve fibers and was discovered by Stoda et al in 1995.The study of NRP1 began with the development of the initial nervous system and has now expanded to include angiogenesis,tumorigenesis and development,hematopoietic diseases,and immune function.NRP1is expressed on almost all tumor cells.NRP1 is expressed on both tumor vascular endothelial cells and tumor cells,so it can play a role in both the tumor microenvironment and tumor cells.At present,NRP1 is highly expressed in lung cancer,prostate cancer,breast cancer,colon cancer,skin cancer,ovarian cancer and other cancer tissues.The survival rate of non-small cell lung cancer patients overexpressing NRP1 is significantly reduced,and the high expression of NRP1 and lung cancer.The poor prognosis of patients is closely related.Current research shows that NRP1 can inhibit tumor cell growth and tumor angiogenesis occurrence and development by inhibiting the expression of NRP1?such as NRP1 monoclonal antibody,RNAi,NRP1 inhibitor,etc.?.Our research team found that high expression of NRP1 produces radiation resistance of lung cancer cells.EMT refers to the biological process by which epithelial cells are transformed into mesenchymal phenotype cells by a specific procedure.During chronic inflammation,fibrosis,and cancer progression,epithelial cells lose their cell polarity,lose their epithelial phenotype,and gain higher migration and invasion,anti-apoptosis,and ability to degrade extracellular matrices.In the iso-phenotype,epithelial cell morphology changes from cobblestone to spindle,showing similarity to fibroblast function after EMT.Epithelial cells undergo EMT in the presence of certain inflammatory cytokines,which in turn promotes the development and progression of fibrosis.Radiation therapy is usually used for the treatment of malignant tumors of the chest,but complications such as IR-induced pulmonary fibrosis or distant metastasis after radiotherapy lead to failure of radiotherapy.Based on previous studies,we studied the expression changes of EMT markers and transcription factors after a single 10 Gy X-ray radiation by studying two lung cancer cell lines with different expression levels of NRP1,and explored the IR-induced lung cancer cells by NRP1.The mechanism of TGF-?/Smads and non-Smads signaling pathways in EMT occurred,and the effect of NRP1 on the migration and invasion of two lung cancer cell lines were further observed.The mechanism of NRP1 on IR-induced EMT in lung cancer cells were elucidated.Purposes:1.To observe the changes of cell morphology,EMT-related transcription factors and markers after X-ray radiation in lung cancer cell models with different expression levels of NRP1;2.To elucidate whether NRP1 passes TGF-?1/Smads and non-Smads signaling pathways in IR-induced EMT;3.To explore the effect of NRP1 on IR-induced migration and invasion of lung cancer cells and its mechanism;Methods:1.Transfection of RetroQ-shNRP1 and pLNCX2-NRP1 into A549 and SK-MES-1 cells by lentiviral transfection to construct NRP1^low-A549,NRP1^high-A549,NRP1^low-SK-MES-1 and NRP1^high-SK-MES-1 cell model;The qPCR and Western Blot methods were used to verify the success of the model establishment;2.Immunofluorescence was used to observe the expression of cytoskeleton?F-actin?in each model group after 10 Gy X-ray radiation.3.The qPCR was used to detect the changes of mRNA time course of EMT markers and transcription factors in the cell models after IR,and to detect the mRNA expression changes of TGF-?1/Smads and non-Smads signaling pathways.4.The Western Blot method was used to detect the time-course changes of EMT markers and transcription factor proteins in the cell models after IR,and to detect the expression changes of TGF-?1/Smads and non-Smads signaling pathway-related molecules;5.Transwell method was used to observe the invasive ability of model cells after IR;6.Using the scratch test method to observe the migration ability of the model cells after IR;7.The time course of CXCL-12 and TGF-?1 in the supernatant of model cells after IR was detected by enzyme-linked immunosorbent assay?ELISA?.Results:1.Radiosensitivity and cell morphology changes of wild-type A549 and SK-MES-1 cellsFirst,cell colony formation experiments were used to observe the survival scores of two lung cancer cells?A549 and SK-MES-1?after exposure to detect the IR sensitivity of different types of lung cancer cells.The results showed that the number of cell colonies decreased significantly after IR in SK-MES-1 cells,and the number of A549 cells were more than that of SK-MES-1 cells at the same IR dose;The survival score of SK-MES-1 were decreased significantly.The survival score of A549 at 2 Gy was 0.636,while the survival score of SK-MES-1 cells were 0.322.It was concluded that SK-MES-1 cells were more sensitive than A549 cells.Then,the changes of cell morphology?F-actin?in wild-type lung cancer cells were observed.It was found that the expression of F-actin in A549 and SK-MES-1 cytoskeleton was enhanced after IR.Finally,the expression of NRP1 in wild-type lung cancer cells were observed.The mRNA and protein levels of NRP1 were significantly increased after IR in A549 cells?P<0.01?.The expression of NRP1 at mRNA level and protein level after IR in SK-MES-1 cell is not significant.2.Establish various cell modelsNRP1 inhibitory plasmid?RetroQ-NRP1?and overexpression plasmid?pLNCX2-NRP1?were transfected into A549 and SK-MES-1 cells by lentiviral transfection method,and each was detected by qPCR and Western blot.Changes in mRNA and protein levels of NRP1 in a panel of cell models.The results showed that NRP1 in NRP1^low-A549 and NRP1^low-SK-MES-1 groups decreased significantly in mRNA and protein levels compared with Vector-A549 group?P<0.01?.The mRNA and protein levels of NRP1 in NRP1^high-A549 and NRP1^high-SK-MES-1 cells were significantly increased?P<0.01?,indicating that the A549 and SK-MES-1 cell models with different expression levels of NRP1 were successfully constructed.3.NRP1 expression and morphological changes of EMT markers in lung cancer cells induced by IR?1?Effect of NRP1 on EMT markers in IR-induced lung cancer cellsAfter IR of the NRP1^low-A549 and NRP1^high-A549 cell models,time course changes of EMT markers??-catenin,N-cadherin and Vimentin?were examined.The results showed that the expression of?-catenin mRNA was increased by 1.72 times?P<0.01?,and Vimentin and N-cadherin were compared with those exposed to light after 0 h.The expression of mRNA at 48 h decreased by 0.53 times and 0.6 times?P<0.01?in the NRP1 low expression group.The expression of?-catenin was not significantly higher in the NRP1 high expression group than in the 0 h group.The mRNA expression of Vimentin and N-cadherin increased by 1.26 times and 1.64times?P<0.01?at 24 h after IR.Similarly,at the protein level of lung adenocarcinoma A549 model group,the expression of?-catenin protein was increased and the expression of N-cadherin protein was decreased in NRP1 low expression after 48 h.The expression of?-catenin protein was decreased and the expression of N-cadherin protein was increased in NRP1 high expression group after 48 h.The time-course changes of EMT markers??-catenin,N-cadherin and Vimentin?were detected after 10 Gy IR of the constructed NRP1^low-SK-MES-1 and NRP1^high-SK-MES-1 cell models.In the lung squamous cell carcinoma SK-MES-1model group,the expression of?-catenin mRNA decreased 0.22 times?P<0.01?in NRP1 low group compared with 0 h after 48 h,and Vimentin and N-cadherin expression decreased by 0.16 times and 0.5 times?P<0.01?;The expression of?-catenin was decreased by 0.1 times?P<0.01?,and the expression of Vimentin and N-cadherin mRNA were not significantly increased after 48 h in high expression of NRP1.Secondly,the expression of?-catenin was increased in the NRP1^low-SK-MES-1group at the protein level compared with 0 h after 48 h,and the expression of Vimentin and N-cadherin protein decreased at 24 h after IR.However,the expression of?-catenin and Vimentin protein were not significantly changed in NRP1^high-SK-MES-1 group compared with 0 h,and only N-cadherin protein expression was elevated.It is suggested that NRP1 may promote IR-induced EMT in A549?adenocarcinoma?cells with higher radiation resistance,while NRP1 may induce IR-induced EMT in SK-MES-1?squamous cell carcinoma?cells with higher radiation sensitivity.?2?Changes in cytoskeleton after IR of lung cancer cell model with different expression levels of NRP1The NRP1^low-A549 and NRP1^high-A549 cell models were stained for 0,12,24and 48 h after a single 10 Gy IR using immunofluorescence.The results showed that NRP1^high-A549 cells were denser than the 0 h group at 48 h after IR,and the expression of the skeleton protein F-actin was increased,showing the spindle filament;The cytoskeletal protein of NRP1^low-A549 cells were not significantly changed at 48 h and 0 h after IR.Although the expression of the skeleton protein F-actin was increased after IR of wild type A549 cells,the expression of the skeleton protein F-actin was weaker than the NRP1^high-A549 group.It is suggested that in IR-induced lung cancer cells,NRP1 may express stromal cell-like morphological changes in lung cancer cells through the expression of cytoskeletal protein F-actin,which may play a role in IR-induced EMT.?3?Effect of NRP1 on EMT-related transcription factors in lung cancer cells induced by IRThe time-course changes of EMT-related transcription factors?ZEB1 and Twist?were examined after 10 Gy IR of the constructed NRP1^low-A549 and NRP1^high-A549model cells.The results showed that the expression of ZEB1 mRNA was not significantly increased in NRP1^low-A549,and the expression of ZEB1 mRNA was decreased by 0.33 times after IR?P<0.01?.The expression of ZEB1 mRNA was increased 1.74 times?P<0.01?at 24 h in NRP1 overexpression group,and the expression of Twist mRNA was not significant.Secondly,the expression of ZEB1 protein was not significantly correlated in the NRP1^low-A549 compared with 0 h,and the Twist protein level was decreased at 48 h after IR.When NRP1 was overexpressed,the expression of ZEB1 protein was increased at 48 h after IR,and the expression of Twist protein was not significant.The time-course changes of EMT-related transcription factors?ZEB1 and Twist?were detected after 10 Gy IR of the NRP1^low-SK-MES-1 and NRP1^high-SK-MES-1model cells.The results showed that the expression of ZEB1 mRNA decreased by 0.6times?P<0.01?after 48 hours of NRP1^low-SK-MES-1.The Twist expression of mRNA level decreased by 0.11 times at 48 h after IR?P<0.01?.The expression of ZEB1mRNA was increased by 1.68 times?P<0.01?at 12 h in NRP1 overexpression,and the expression of Twist mRNA was increased by 2.47 times at 24 h after IR?P<0.01?.Secondly,in the NRP1^low-SK-MES-1 the expression of ZEB1 and Twist decreased at 48 h after IR.When NRP1 was overexpressed,it was compared with 0h,the expression levels of ZEB1 and Twist protein were elevated at 48 h.It is suggested that the low expression of NRP1 in the radiation-resistant A549cell group and the radiation-sensitive SK-MES-1 cell group can inhibit the expression of EMT-related transcription factors induced by IR.High expression of NRP1promotes the expression of EMT-related transcription factors induced by IR.4.Effect of NRP1 on EMT-related signaling pathways in lung cancer cells induced by IR?1?Effect of NRP1 on TGF-?1/Smads signaling pathway in lung cancer cells induced by IRThe expression of TGF-?1 in lung adenocarcinoma cells were detected by ELISA and qPCR.The expression of TGF-?1 mRNA in NRP1^low-A549 cells were not significant compared with at 0 h.NRP1^low–A549 in cell culture supernatant was detected.The secretion of TGF-?1 was decreased in the cells treated for 24 h?P<0.01?;The expression of TGF-?1 mRNA was significantly increased in NRP1^high-A549 cells?P<0.01?.The secretion of TGF-?1 in NRP1^high-A549 cells were increased for 12 h?P<0.01?.The expression of Smads-related signaling molecules in lung adenocarcinoma cells were detected by WB and qPCR.The expression of Smad2and Smad3 in NRP1^low-A549 cells were not significantly higher than that at 0 h.The expression of Smad2 and Smad3 in NRP1^high-A549 group were increased by 2.45times and 1.37 times?P<0.01?after 12 h.Secondly,the changes of Smads protein levels were observed.The expression of Smad3 protein was significantly decreased in NRP1^low-A549 cells compared with 0 h,but the expression of Smad2 and Smad3 were significantly increased in NRP1^high-A549 cells 48 h after IR.The expression of TGF-?1 in lung squamous cell carcinoma cells were detected by ELISA and qPCR.The expression of TGF-?1 mRNA was increased in NRP1^low-SK-MES-1 group compared with 0 h?P<0.01?.In the cell culture supernatant,the secretion of TGF-?1 was decreased in the NRP1^low-SK-MES-1 group for 24 h?P<0.01?.The expression of TGF-?1 was significantly increased in the NRP1^high-SK-MES-1 group compared with 0 h?P<0.01?.In the NRP1^high-SK-MES-1group the secretion of TGF-?1 was increased at 12 h?P<0.01?.The expression of Smads-related signaling molecules in lung squamous cell carcinoma cells were detected by WB and qPCR.The mRNA level expression of Smad2 and Smad3 in NRP1^low-SK-MES-1 cells were decreased at 48 h after IR?P<0.01?;However,Smad2 and Smad3 were not significantly different in the NRP1^high-SK-MES-1 group compared with 0 h.Secondly,the changes of Smads protein levels were observed.The expression of Smad2 and Smad3 protein were significantly decreased in NRP1^low-A549 cells compared with 0 h,but the expression of Smad2 and Smad3 in NRP1^high-A549 cells were not significantly higher than in 0 h.It is suggested that the low expression of NRP1 in A549 cells with high radiation resistance can inhibit the activation of TGF-?1/Smads signaling pathway,and the high expression of NRP1 can enhance the activity of TGF-?1/Smads pathway after IR.In the SK-MES-1 cell group with high radiation sensitivity,the low expression of NRP1inhibited the activation of TGF-?1/Smads pathway,while the high expression of NRP1 did not significantly activate the TGF-?1/Smads pathway.?2?Effect of NRP1 on IR-induced non-Smads signaling pathway in lung cancer cellsThe expression of non-Smads-related signaling proteins in lung adenocarcinoma cells were detected by WB and qPCR.The expression of Akt and NF-?B mRNA were decreased in NRP1^low-A549 cells compared with 0 h.The expression of Akt and NF-?B mRNA were increased in NRP1^high-A549 cells after 24 h.At the protein level,the expression of p-Akt and NF-?B protein were increased after 24 h.The expression of Akt,p-Akt and NF-?B protein in NRP1^low-A549 group and NRP1^high-A549 group were not significant compared with 0 h.In addition,the expressions of NF-?B-related signaling molecules IKK?and I?B-?were detected.The expression of IKK?and I?B-?mRNA in NRP1^low-A549cells were not significantly different after 0 h.The expression of IKK?and I?B-?mRNA were increased in the NRP1^high-A549 group at 48 h after IR.At the protein level,the expression of IKK?protein was increased in wild-type A549 cells after 24 h,but the expression of IKK?and I?B-?protein in NRP1^low-A549 and NRP1^high-A549cells were not significantly higher than that in 0 h.The expression of non-Smads-related signaling proteins in lung squamous cell carcinoma cells were detected by WB and qPCR after IR.The expression of Akt and NF-?B mRNA in NRP1^low-SK-MES-1 group were decreased after 48 h compared with 0 h?P<0.01?.The expression of Akt and NF-?B mRNA in NRP1^high-SK-MES-1group were not significantly increased after IR;At the protein level,the expression levels of Akt,p-Akt and NF-?B proteins were increased after IR to wild-type SK-MES-1 cells.The expression of Akt,p-Akt and NF-?B protein in NRP1^low-SK-MES-1 group and NRP1^high-SK-MES-1 group were not significant after IR.In addition,the expression of NF-?B-related signaling molecules IKK?and I?B-?were detected.The expression of IKK?mRNA in NRP1^low-SK-MES-1 cells was decreased after 48 h compared with 0 h?P<0.01?.The expression of I?B-?mRNA was increased in NRP1^high-SK-MES-1 compared with 0 h?P<0.01?.At the protein level,the expression of IKK?protein was increased at 12 h after IR to wild-typeSK-MES-1cells,whilethatinNRP1^low-SK-MES-1and NRP1^high-SK-MES-1 cells IKK?and I?B-?protein expression were not significant compared with 0 h.It is suggested that the wild-type A549 and SK-MES-1 cells have significant changes in non-Smads signaling pathways after IR.However,the non-Smads signaling pathways were not significantly changed in the two lung cancer cell models with different expression levels of NRP1.It is indicated that the regulation of NRP1 on non-Smads pathway protein levels in A549 and SK-MES-1 cells is not significant.5.Effects of NRP1 on the migration and invasion of lung cancer cells after exposureIt is known from the above results that the effect of NRP1 in IR-induced lung cancer cell line EMT is greater than that of squamous cell carcinoma?SK-MES-1?.Therefore,the effect of NRP1 on the migration and invasion of lung adenocarcinoma were emphasized.After the NRP1^low-A549 and NRP1^high-A549model cells were radiated by 10 Gy X-ray,the effect of NRP1 on the invasion ability of lung adenocarcinoma cells were observed by Transwell method.The results showed that the number of invasive cells in the NRP1 high expression group were significantly increased after 48 h compared with 0 h?P<0.05?.However,the number of invasive cells in the NRP1 low expression group were not significantly increased after 48 h compared with 0 h.It is suggested that NRP1 enhances the ability of radiation to induce migration and invasion of lung cancer cells.Scratch method was used to observe the migration ability of lung adenocarcinoma cells with different expression levels of NRP1.The results showed that the migration ability of NRP1 overexpression group was significantly increased after 48 h exposure compared with 0 h?P<0.05?,while the migration ability of NRP1low expression group was not significant.The expression of CXCL-12 in the culture supernatants of NRP1^low-A549 and NRP1^high-A549 cells were detected by ELISA.The results showed that the CXCL-12secretion increased in the NRP1 high expression group after 12 h compared with 0 h?P<0.01?,and the NRP1 low expression group was exposed to CXCL-12 after 48h,the amount decreased?P<0.05?.It is suggested that the secretion of tumor metastasis marker CXCL-12 is increased,indicating that NRP1 can promote IR-induced lung cancer cell migration.Conclusion:1.IR can induce EMT in lung adenocarcinoma cells?A549?.In the process,NRP1 can promote the emergence of spindle filaments in lung cancer cells induced by IR,and the expression of F-actin is enhanced.2.NRP1 promotes the occurrence of EMT in lung cancer cells induced by IR,that is,the expression of epithelial markers is decreased,and the expression of mesenchymal markers is increased,which may be related to the increased expression of EMT-related transcription factors;3.NRP1 may promote the IR-induced EMT of lung cancer adenocarcinoma cells mainly through TGF-?1/Smads signaling pathway;4.NRP1 promotes the EMT of lung cancer cells,and enhances the invasion and migration ability of lung adenocarcinoma cancer cells induced by IR.