| Objective:Lung cancer is a highly aggressive,highly recurrent malignant tumor with high mortality and poor prognosis,with a 5-year overall survival rate of less than 20%.Tumor metastasis is the leading cause of death in lung cancer patients.Epithelial-mesenchymal transtition(EMT)is one of the important mechanisms of metastasis of tumor cells.After the occurrence of EMT,tumor cells gain a stronger ability to invade and migrate.EMT also imparts a stem cell-like phenotype to tumor cells,i.e.,stemness.Both EMT and stemness induce chemoresistance in tumor cells.The poliovirus receptor(PVR),commonly known as CD155,is highly expressed in a variety of tumor cells and has been implicated in poor prognosis in patients,but the role of CD155 in lung cancer and its associated mechanisms remain unclear.The purpose of this study was to explore the role of CD155 in the development and development of lung cancer,especially to analyze the effects of CD155 on EMT,stemness and chemotherapy resistance of lung cancer cells and its mediated signaling pathways,so as to provide new strategiesMethods:1.Analysis of CD155 expression in lung cancer tumor tissues and its correlation with the prognosis of lung cancer patients through the Kaplan-Meier Plotter website.2.The CCLE database was used to analyze the correlation between CD155 and interstitial phenotype and stem-related indexes.3.Detection of CD155 expression in clinical tissue samples of lung cancer: 30 clinical samples confirmed by histopathology as lung cancer and 10 cases of adjacent tissue samples were selected.The expression of CD155 in the sample was detected by immunohistochemistry.4.To explore the EMT regulatory effect of CD155 on lung cancer A549 and H1299 cells: CD155 sh RNA lentivirus transfected A549 and H1299 cells,and the knockdown efficiency was confirmed by Real-time PCR,Western blot experiment and cellular immunofluorescence technology.The correlation between CD155 and tumor cell interstitial phenotypic indexes VIM,FN1,TGFB1 and SMAD3 was analyzed in CCLE database.Real-time PCR assays detected the expressionof CD155-reduced indexes VIM,FN1,ACTA2 and CDH1 m RNA related indicators related to interstitial phenotype.The expression of E-cadherin,Vimentin,Fibronectin and α-SMA in CD155 knocked cells was detected by cellular immunofluorescence technology.The morphological changes of A549 and H1299 cytoskeleton were detected by the clinopeptide staining experiment,and their effects on the interstitial phenotype of tumor cells were judged.5.To explore the EMT regulatory effect of CD155 on LK2 cells of lung cancer: CD155 overexpressed lentiviral transfection of LK2 cells,and the gene overexpression efficiency was confirmed by Real-time PCR,Western blot experiment and cellular immunofluorescence technology.The expression of E-cadherin and Vimentin in cells after CD155 knockdown was detected by cellular immunofluorescence technology.The morphological changes of LK2 cytoskeleton were detected by the ghostin staining experiment,and its effect on the interstitial phenotype of tumor cells was judged.6.Analysis of the effect of CD155 on the migration and invasion ability of lung cancer cells: In the stable transition knockdown(A549-sh CD155,H1299-sh CD155)and overexpression(LK2-CD155oe)stable cell lines,the effect of CD155 on the migration and invasion ability of lung cancer cells was detected by scratch experiment and Transwell invasion experiment.The tail vein of immunodeficient mice was injected with CD155 to knock down the lung cancer cell line,the number of lung metastases was observed and counted,the size of lung tissue metastases was observed by HE staining,and the effect of CD155 on tumor migration and metastasis was detected in vivo and in vitro experiments.7.To explore the effect of CD155 on stemness of lung cancer cells: the expression of CD44 m RNA,an index related to stemness,was detected by Real-time PCR experiments in the cell line transfected with CD155 lentivirus;The expression of CD44 in tumor cells was detected by flow cytometry;In vitro 3D spheroidization experiments were used to detect the spheroidic ability of tumor cells after interfering with or overexpressing CD155,and to judge the effect of CD155 on the maintenance of stem ability of lung cancer cells.8.To explore the effect of CD155-mediated lung cancer cells on the sensitivity of chemotherapy drugs: the sensitivity of CD155 stable knockdown or overexpression cell lines to cisplatin(Cisplatin)was detected by CCK-8 experiment;Flow cytometry PI/Annexvin V double stain was used to detect the apoptosis ratio of stable cell lines.In vitro 3D spheroidization experiments were used to detecttumor cells knockdown or overexpression of CD155,and cisplatin chemotherapy was given to stimulate the spheroidation ability of cells and determine the effect of CD155 on the resistance of chemotherapy drugs in lung cancer.9.Detection of the regulatory effect of CD155 on YAP1: the correlation between CD155 and YAP1 in lung cancer cells was analyzed by CCLE database,and the expression of YAP1 in lung cancer cells was detected by Real-time PCR experiment and Western blot experimental technology.10.Exploring the reversal of chemotherapy-resistant phenotypic changes in sh CD155 lung cancer cells by YAP1 overexpression plasmid: The expression of YAP1 in transfected cells was detected by Real-time PCR experiment and Western blot experiment: the change of cell viability of YAP1 overexpressed cells at 0,2.5,5,10 and 20μM concentrations of cisplatin was detected by CCK-8 experiment.Cell survival curves were plotted to evaluate the effect of YAP1 on the sensitivity of cellular chemotherapy.Flow cytometry PI/Annexvin V double stain was used to detect the apoptosis ratio of transfected cells.In vitro 3D spheroidization assay was used to detect the stemness maintenance ability of cisplatin-treated YAP1 overexpressed cells.11.The results of the YAP1 overexpression plasmid transfection CD155 knockdown stable transfer cells,the results of the phalloidin staining experiment showed that the morphology of tumor cells was transformed from epithelioid to interstitial mode,and the migration and invasion ability of lung cancer cells was enhanced by scratch experiment and Transwell invasion experiment.12.Exploring the regulation of EMP3 by YAP1: the CCLE database was used to analyze the correlation between EMP3 and CD155,EMP3 and YAP1 in lung cancer cell lines;Real-time PCR and Western blot experiments were used to detect the expression changes of EMP3 m RNA and protein in lung cancer cells after CD155 knockdown.Overexpression of YAP1 in strophony cells of sh CD155 lung cancer,detection of expression changes of EMP3 m RNA and protein by Real-time PCR experiment and Western blot experiment.Ch IP experimental technology was used to detect the binding interaction between YAP1 and EMP3 promoter regions.13.Analysis of EMP3 overexpression to reverse the phenotypic changes of chemotherapy resistance in sh CD155 lung cancer cells: EMP3 overexpression plasmids were used to transfect CD155 knockdown stable transfusion cells,and the expression of EMP3 in transfected cells was detected by Real-time PCR experiment and Western blot experiment: throughCCK-8 experiment,the change of cell viability of EMP3 overexpressed cells under the action of cisplatin concentration of 0,2.5,5,10 and 20 μM was detected.Cell survival curves were plotted to evaluate the effect of EMP3 on cellular chemotherapy sensitivity.Flow cytometry PI/Annexvin V double staining was used to detect the apoptosis ratio of cells after treatment.In vitro 3D spheroidization assays were used to detect the stemness maintenance ability of cisplatin-treated EMP3 overexpression cells.14.Exploration of EMP3 overexpression to reverse sh CD155 mesenchymal phenotypic changes in lung cancer cells: EMP3 overexpression plasmids were used to transfect CD155 knockdown stable transfer cells,and the immunofluorescence staining experiment of phalloidin was used to detect the morphological changes of the skeleton of lung cancer cells;Scratch assay and Transwell invasion assay were used to detect changes in EMT-mediated cell migration and invasion capacity in lung cancer cells.Results:1.Analysis through the Kaplan-Meier Plotter website showed that CD155 was associated with overall survival(OS),first progression survival(FP)and post-progression survival(PPS)in lung cancer patients,and the results showed that CD155 high expression patients had a poor prognosis.2.CCLE database analysis showed that CD155 in lung cancer cell lines was positively correlated with tumor interstitial and stemness indexes.3.By immunohistochemical detection method,30 lung cancer tissue samples and 10 cases of paracancerous tissue were detected,and it was found that the expression of CD155 in tumor tissues was higher than that in the adjacent cancer control group.4.CD155 sh RNA lentivirus transfection of A549 and H1299 cells,Real-time PCR,Western blot experiment and cellular immunofluorescence technology confirmed the CD155 interference efficiency.Compared with the control group,the expression of CDH1 in cells after CD155 knockdown was significantly increased,and the expression of VIM,FN1 and ACTA2 were reduced in the interstitial phenotype-related indexes.Immunofluorescence technology detected an increase in E-cadherin expression and a significant decrease in the expression of Vimentin,Fibronectin and α-SMA in CD155-reduced cells.The staining of the clinopeptide of the Pseudocyclic Peptide detected the morphological transformation of the cytoskeleton of A549 and H1299 from the interstitial to the epithelium.5.CD155 overexpression plasmid transfection of lung cancer LK2 cells,Real-time PCR,Western blot experiment and cellular immunofluorescence technology confirmed the overexpression efficiency of CD155.Compared with the control group,the expression of CDH1 in cells after CD155 overexpression was significantly reduced,and the expression of VIM,FN1 and ACTA2 were increased in the interstitial phenotype-related indexes.Immunofluorescence technology detected a decrease in E-cadherin expression and a significant increase in the expression of Vimentin,Fibronectin and α-SMA in cells after CD155 overexpression.The staining experiment of Gyrobin Cyclic Peptide detected the morphological transformation of LK2 cytoskeleton from epithelium to interstitium.6.In CD155 stable knockdown(A549-sh CD155,H1299-sh CD155)and overexpression(LK2-CD155oe)stable cell lines,scratch experiment and Transwell invasion experiment verified the ability of CD155 to promote lung cancer cell migration and invasion.Compared with the control group,the number of lung cancer metastases and the volume of cancer nodules in the CD155 knockdown group were significantly reduced.7.In CD155 knockdown stable cell line,Real-time PCR experiments detected a decrease in the expression of CD44 m RNA,a stem-related index.The proportion of CD44 expression detected by flow cytometry was reduced;Using in vitro 3D spheroidic experiments,it was found that tumor cells had a decrease in their ability to knock down CD155 spheroids,and conversely,after overexpressing CD155,the spheroid-forming ability of cells was enhanced.8.Through CCK-8 experiments,the sensitivity of CD155 stable knockdown cells to cisplatin(Cisplatin)was enhanced compared with the control group;The proportion of apoptosis in stable cells using flow cytometry was significantly increased;In vitro 3D spheroidization experiments confirmed that after tumor cells knocked down CD155,cisplatin chemotherapy was given stimulation,the spherogenic ability of tumor cells decreased.The above results were further verified by CD155 overexpression cell lines,and it was found that CD155 promoted tumor cell drug resistance,inhibited the proportion of apoptotic cells,and improved tumor cell spheroidy.9.CCLE database analysis confirmed that CD155 and YAP1 were positively correlated in a series of lung cancer cells,and CD155 in CD155 knockdown and overexpression of CD155 in lung cancer cells was detected by Real-time PCR experiment and Western blot experimental technology to have a positive regulatory relationship between CD155 protein expression in lung cancercells.10.CD155 knockdown overexpression of YAP1 in stable cells,CCK-8 experimental results showed that tumor cell activity changes were enhanced under different concentrations of cisplatin drug treatment,suggesting that YAP1 promoted tumor cell chemotherapy resistance;The results of flow cytometry PI/Annexvin V double stain showed that YAP1 overexpression inhibited the apoptosis ratio of stable cells.In vitro 3D spheroidization experiments confirmed that YAP1 overexpressed cells had a stronger ability to maintain stemness after cisplatin treatment than the control group.11.The results of the YAP1 overexpression plasmid transfection CD155 knockdown stable transfer cells,the results of the phalloidin staining experiment showed that the morphology of tumor cells was transformed from epithelioid to interstitial mode,and the migration and invasion ability of lung cancer cells was enhanced by scratch experiment and Transwell invasion experiment.12.CCLE database analysis showed a significant positive correlation between EMP3 and CD155,EMP3 and YAP1 in lung cancer cell lines.The expression levels of EMP3 m RNA and protein in lung cancer cells decreased after the detection of sh CD155 by Real-time PCR and Western blot experiment.Overexpression of YAP1 in sh CD155 lung cancer cells,Real-time PCR experiments and Western blot experiments confirmed elevated expression of EMP3 m RNA and protein;Using Ch IP experimental technology,it was confirmed that YAP1 bound to the CTCATTCCTCCA sequence in the EMP3 promoter region.13.Overexpression of EMP3 in stable transgenic cells of sh CD155,Real-time PCR experiment and Western blot experiment detected the upregulation of EMP3 expression in transfected cells.The results of CCK-8 showed that tumor cell activity was enhanced under different concentrations of cisplatin,suggesting that EMP3 promoted tumor cell chemotherapy resistance.The results of flow cytometry PI/Annexvin V double staining showed that EMP3 overexpression inhibited the apoptosis ratio of stable cells.In vitro 3D spheroidization experiments confirmed that EMP3 overexpressed cells maintained their dryness after cisplatin treatment with a stronger ability than the control group.14.EMP3 overexpression plasmid transfected CD155 knockdown stable transfer cells,the results of the clinopeptide staining experiment showed that tumor cells were transformed from epithelioid to interstitial mode,and the scratch experiment and Transwell invasion experiment detected the migration and invasion ability of lung cancer cells.Conclusions:1.Increased expression of CD155 in lung cancer tissues is associated with poor prognosis of patients.2.CD155 promotes the migration and invasion of lung cancer cells,maintains stem cell-like characteristics of tumor cells,and induces chemotherapy resistance in lung cancer cells.3.CD155 promotes the interstitial phenotype and stem-like characteristics of lung cancer cells and induces chemotherapy resistance in lung cancer cells by regulating YAP1.4.YAP1 regulates EMP3 through transcription,promotes the interstitial phenotype and stem-like characteristics of lung cancer cells and induces chemotherapy resistance in lung cancer cells.5.CD155-YAP1-EMP3 signaling pathway can be used as a potential therapeutic target for lung cancer metastasis and drug resistance. |
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