Effects Of YAP/NRP1 On Radiation Resistance Of Lung Cancer Cells And The Regulatory Mechanism

Lung cancer is one of the most commonly diagnosed cancers worldwide and the leading cause of cancer-related deaths,and China is a country with a high prevalence of lung cancer,which has become a serious threat to human life and health.Among them,non-small cell lung cancer(NSCLC)is the most common histological type of lung cancer,accounting for more than 85%of the cases.Among the classifications of NSCLC,lung adenocarcinoma is the most common subtype.There are various means to treat lung cancer,including surgical resection,chemotherapy,radiation therapy,thermotherapy,immunotherapy,etc.Among them,radiation therapy is the most important clinical treatment.Among them,radiation therapy,as one of the most prominent clinical lung cancer treatments,can significantly improve the local control rate of tumor and patient survival.However,the phenomenon of tumor recurrence or metastasis due to radiation resistance of tumor cells after repeated irradiation still exists.Therefore,studying the molecular mechanisms underlying the development of radiation resistance in lung cancer cells and finding new therapeutic strategies are crucial to improve patient prognosis.Background:NRP1 is a transmembrane glycoprotein that acts as an oncogene and is involved in the development of various malignancies,such as lung,liver,breast and colorectal cancers and bladder cancer,among others.Previous studies by our group have shown that NRPl is highly expressed in non-small cell lung cancer and promotes the epithelialmesenchymal transition(EMT)process in lung cancer cells by interacting with TGF-β,which in turn enhances the invasion and metastasis of lung cancer cells.Therefore,NRP1 gene is closely related to the radioresistance of lung cancer cells.YAP is a downstream factor of Hippo pathway,which controls cell proliferation and apoptosis by regulating DNA replication,DNA repair and transcription of genes related to apoptosis.YAP is upregulated in a variety of cancer cells,including hepatocellular carcinoma,glioma and colorectal cancer,etc.High expression of YAP or activation of its pathway is closely associated with poor prognosis of malignant tumors.Therefore,YAP is an important therapeutic target in cancer.Our group has demonstrated that NRP1 plays an important role in the generation of radiation resistance in lung cancer cells,and YAP has been reported to be significantly overexpressed in non-small cell lung cancer.In this study,we investigated the regulatory relationship between YAP and NRP1 in parental and radiation-resistant cells and the effect and regulatory mechanism of YAP/NRP1 on radiation resistance,aiming to lay the foundation and provide new experimental basis for improving the efficacy of tumor radiotherapy and opening up new therapeutic strategies.Objective:In this study,we intend to establish a radiation-resistant cell model using two lung adenocarcinoma cell lines,A549 and H1299,to investigate the regulatory effects of YAP on the transcriptional and translational levels of NRP1,to analyze the regulatory patterns in parental cells and lung adenocarcinoma radiation-resistant cells,and to explore the effects of YAP/NRP1 on radiation resistance in lung cancer cells,aiming to provide new targets and new ideas for clinical lung cancer radiotherapy regimens.Methods:1.Radiation resistance(RR)cell models,named A549-RR and H1299-RR cells,were established using a high-dose fractionated irradiation method.Two types of lung adenocarcinoma A549 and H1299 cells were irradiated with 6 Gy each time at dose rates of 1.02 Gy/min and 0.75 Gy/min,respectively,for a total of 5 times and a total irradiation dose of 30 Gy.The radiation-resistant cell models were characterized by clone formation assay,cell proliferation assay,cell morphology photography and flow cytometry.2.The migration ability of parental cells and radioresistant cell models was examined by scratch assay.3.To detect the invasion ability of parental cells with the radiation resistant cell model by cell invasion assay.4.To detect changes in mRNA expression of NRP1 and YAP by qRT-PCR.To detect changes in mRNA expression of NRP1 by knocking down or overexpressing YAP in parental cells and radiation-resistant cell models.5.Detection of protein expression changes of NRP1 and YAP by Western Blot;protein expression of YAP in the cytoplasm and nucleus of cells;knockdown or overexpression of YAP in parental cells and radiation-resistant cell models,and detection of protein expression changes of NRP1.6.To observe the intracellular localization of YAP in parental and radiationresistant cell models by immunofluorescence staining.7.To construct YAP overexpression plasmids by molecular biology techniques(genetic engineering)by modifying the existing null-loaded plasmid pCDH-CMVMCS-EF1-CopGFP-T2A-Puro in the group,and send them to Coomassie for sequencing verification.8.By transient transfection assay,the overexpression or knockdown YAP plasmids were transfected into parental cells and radioresistant cell models,and the success of transfection was verified by qRT-PCR and Western Blot.Results:1.Establishment of radiation-resistant cell model for lung adenocarcinoma and its identification(1)Establishment of radiation-resistant cell models for lung adenocarcinomaRadiation-resistant cell models were constructed for A549 and H1299 cells by fractional irradiation at dose rates of 1.02 Gy/min and 0.75 Gy/min,with 6 Gy each time and 5 repetitions for a total dose of 30 Gy.After completion of the models,the 2 cells were named A549-RR and H1299-RR two Identification of radioresistant cell modelsThe results showed that the number of colonies formed by the radioresistant cell model was significantly higher than that of the parental cells when irradiated with the same dose,indicating that the radiosensitivity of both A549-RR and H1299-RR cells was significantly decreased.In addition,the survival fraction of A549 at 2 Gy was 0.485,while that of A549-RR cells was 0.733,and that of H1299 at 2 Gy was 0.514,while that of H1299-RR cells was 0.811.Compared with the parental cells,the D0 value of the radioresistant cell model decreased while the Dq value increased significantly,indicating that the radioresistant cell model The stronger the ability to repair sublethal damage,the weaker the cellular radiosensitivity.Therefore,the results of the clone formation experiment indicated that the radiation resistance of the radioresistant cell model was higher than that of the parental cells,which proved that the radioresistant model cells were successfully established.Optical microscopy observed that the parental cells and the radioresistant model cells changed in cell morphology.The results showed that A549-RR cells increased in size and changed from regular shuttle shape to irregular sickle shape,while H1299-RR cells also increased in size and showed obvious morphological changes.The proliferation ability of the parental cells and the radiation-resistant cell model was examined using CCK-8 assay,and the results showed that the survival rate of A549RR and H1299-RR cells was higher under different doses of irradiation.Flow cytometry was used to detect apoptosis after 10 Gy irradiation in the parental and radiation-resistant cell models,and the apoptosis rate was found to be 51.5%(p<0.05)for A549-RR and(p<0.01)for H1299-RR cells,which were significantly lower cells compared with the parental cells.Taken together,the results indicate that the radioresistant cell model was successfully established.2.Changes in NRP1 in a radiation-resistant cell of lung adenocarcinoma and associated phenotypic changes in its effects(1)Changes in NRP1 in the radiation-resistant cell modelThe mRNA expression of NRP1 in the parental and radiation-resistant cell models was detected by qRT-PCR,and NRP1 was elevated 1.6-fold(p<0.01)in A549-RR cells and 11.2-fold(p<0.01)in H1299-RR cells.The WB method was used to detect changes in NRP1 protein levels,which were elevated in both A549-RR and H1299-RR cells compared to A549 and H1299 cells.(2)Changes in migration and invasion in the radiation-resistant cell modelThe changes in cytoskeleton were detected using ghost pencil loop peptide staining,and the results showed that only a small amount of dispersed irregular fibrous network was present in A549 and H1299 cells,and cytoskeletal proteins were significantly increased in the membrane and perinuclear regions in A549-RR and H1299-RR cells.The change of cell migration ability was detected by scratch assay,and the results showed that the migration ability of A549-RR and H1299-RR cells was significantly enhanced.Cytoskeletal proteins(F-actin)are the main adhesion-related proteins between cells and between cells and the matrix,and this adhesion ability is important for regulating cell migration and proliferation.Therefore,when cellular radioresistance is enhanced,the cellular adhesion capacity is altered,which in turn will lead to changes in cell migration and invasion ability.Transwell assay was used to detect the changes of invasion ability of cells,and the results showed that the number of invading cells in the radiation-resistant cell models A549-RR and H1299-RR was significantly increased compared with that of A549 and H1299 cells.This indicates that the invasive ability of the radiation resistant cell model was enhanced.3.Screening and expression changes of radioresistance-related molecules in lung adenocarcinoma radiation resistance cell models(1)Gene expression profiling microarray to search for differential genes in A549 and A549-RR cellsIn this study,gene expression profiling microarray(RNA-Sequence)was used to find differentially expressed genes in A549 and A549-RR cells.The results showed that a total of 15747 genes were expressed in A549 and 15920 genes were expressed in A549-RR cells,and there were 15126 common genes with expression differences between the two.Among these shared genes,there were 3888 up-regulated genes and 4067 down-regulated genes.The heat map of gene differential clustering showed that both NRP1 and YAP were up-regulated genes in A549-RR cells.Prediction of transcription factors upstream of the NRP1 gene using the JASPAR bioinformatics website revealed that there are sites upstream of the NRP1 gene that bind to YAP,and seven of them have a binding rate of 98%or higher.Functional analysis of differential genes(GO)showed that differential genes were mainly enriched in biological processes associated with the cytoplasmic membrane.Differential gene signaling pathway analysis(KEGG)showed that differential genes were mainly concentrated in pathways associated with cancer.Based on the above results,it was shown that YAP and NRP1 are important genes for the formation of radioresistance.(2)Validation of YAP changes in the radioresistant cell modelThe changes in mRNA level expression of YAP in the radioresistant cell model were examined using qRT-PCR assay.The results showed that YAP was elevated 2.2fold in A549-RR cells(p<0.01)and 2.2-fold in H1299-RR cells(p<0.01).Validation of the protein levels of YAP in the radioresistant cell model using WB experiments showed that the protein levels of YAP were significantly elevated in both A549-RR and H1299RR cells compared to A549 and H1299 cells.This is consistent with the microarray results.(3)Changes in YAP protein localization in parental cells and radioresistant cell modelsImmunofluorescence staining was used to detect the intracellular localization of YAP in the parental and radiation-resistant cell models,and the results showed that YAP entry into the nucleus was significantly increased in A549-RR and H1299-RR cells compared with A549 and H1299 cells.The nucleation of YAP was re-validated by WB method.The results showed that WB experiments obtained consistent results with immunofluorescence staining experiments.4.Regulation of YAP and NRP1 in a lung adenocarcinoma radioresistant cell model(1)Construction of YAP overexpression plasmid and validation of plasmid sequenceThe YAP overexpression plasmid was constructed using genetic engineering,and the null plasmid pCDH-CMV-MCS-EF 1-CopGFP-T2A-Puro was modified by inserting a YAP fragment between Xba I and EcoR I restriction endonucleases.Whole RNA from human normal lung epithelial cells(BEAS-2B)was extracted and a large number of YAP fragments were amplified using PCR.The overexpressed YAP plasmid was obtained by double digestion of the YAP fragment of the target gene and the excised null plasmid after T4 ligase ligation,transformation and plasmid miniprep.The synthesized YAP overexpression plasmid was identified using agarose gel electrophoresis,and the results showed that the position of the YAP fragment of the target gene was consistent with the expected results.The recombinant plasmid was also sent to Kumi for sequencing and identification,and the results showed that the plasmid sample sequence matched 100%with the YAP sequence.In conclusion,it was proved that the YAP overexpression plasmid was successfully constructed and named as YAPOE.(2)Changes of NRP1 after overexpression of YAP in parental cells and radiationresistant cell modelsThe YAP OE plasmid was transfected in parental cells and radiation-resistant cell models using transient transfection.The changes in mRNA and protein expression of YAP and NRP1 were detected by qRT-PCR and WB method.The results showed that YAP was elevated to 4.4-fold in A549 cells and 8.2-fold in A549-RR cells after transfection with YAP OE,indicating successful transfection of YAP OE.The change of NRP1 was detected,and NRP1 decreased to 0.8-fold in A549 cells and increased nearly 1.5-fold in A549-RR cells.The results for protein were consistent with those for mRNA.Transfection of YAP OE in H1299 and H1299-RR cells showed that YAP was elevated to 38.8-fold in H1299 cells and 37.1-fold in H1299-RR cells,indicating successful transfection of the overexpression plasmid.The change of NRP1 was detected,and the expression was unchanged in H1299 cells,while it increased 1.7-fold in H1299-RR cells.The results for protein were consistent with those for mRNA.(3)Changes in NRP1 after knockdown of YAP in parental and radioresistant cell modelsYAP knockdown plasmids(pPLK/GFP+Puro-YAP shRNA)were transfected in parental and radiation-resistant cell models using transient transfection,and the changes in mRNA and protein expression of YAP and NRP1 were detected using qRT-PCR and WB method.The results showed that the mRNA level of YAP in A549 and A549-RR cells was reduced to 0.6-fold,which showed that the plasmid transfection was successful.The changes of NRP1 were detected,and the expression of NRP1 was reduced to 0.8-fold in A549 cells and to 0.6-fold in A549-RR cells.The results for protein were consistent with those for mRNA.After transfection of YAP knockdown plasmid in H1299 and H1299-RR cells,the mRNA level of YAP was reduced to 0.3fold in both H1299 and H1299-RR cells,indicating the successful transfection of knockdown plasmid.The changes of NRP1 were detected,and the expression of NRP1 was significantly reduced to 0.1-fold and 0.3-fold in both H1299 and H1299-RR cells,respectively.The results for protein were consistent with those for mRNA.Taken together,YAP as the upstream of NRP1 gene does regulate the transcription and translation of NRP1.5.YAP knockdown or overexpression can affect the phenotypic changes in lung adenocarcinoma radiation-resistant cell modelsClonogenesis assay,CCK-8 assay,scratch assay,and Transwell assay were used to examine the changes in parental cells and the two radioresistant cell models after knockdown or overexpression of YAP.When YAP was overexpressed,the number of colony formation was increased,cell survival was increased,and migration and invasion abilities were enhanced compared to the null group.When YAP was knocked down,the number of colony formation was significantly reduced,cell survival was decreased,and migration and invasion abilities were significantly reduced compared to the NC group.Conclusions:1.NRP1 and YAP expression were significantly increased during the formation of radioresistance in two lung adenocarcinoma cells.2.YAP phosphorylation decreased into the nucleus significantly increased in both lung adenocarcinoma radiation resistant cells.3.YAP was an upstream regulator of NRP1 in both lung adenocarcinoma radiation resistant cells and YAP was positively correlated with NRP1.4.Knockdown of YAP reversed the radioresistance of lung adenocarcinoma cells and improved radiosensitivity.