| Lung cancer is the most common tumor and which has the highest mortality rate worldwide.More than 80% of the lung patients were diagnosed as non-small cell lung cancer(NSCLC).There are two main subtypes of NSCLC: lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC).Different subtypes of NSCLC are related to different molecular biological characteristics.The molecular mechanism of the two subtypes of NSCLC is different,which leads to obvious differences in the treatment plan and clinical response.Therefore,depth analysis of the differential genes between LUSC and LUAD is helpful to find the signal pathway and molecular diagnostic markers that can be used to distinguish and treat LUSC and LUAD.Exosomes play different roles in tumors,such as immune regulation,formation of pre-metastasis niches,tumor growth,regulation of treatment resistance,and drug excretion mediated by exosomes.Because exosomes are closely related to the state of primordial cells,relatively stable in the humoral circulation,and easy to collect from patients,exosomes are ideal biomarkers for tumor diagnosis,prognosis and treatment.However,up to now,it is not clear whether there are ideal molecular diagnostic markers in exosomes of NSCLC patients to distinguish LUSC and LUAD.Therefore,we conducted this study to determine whether the m RNA carried in the patient’s plasma exosomes can effectively distinguish patients with lusc and luad.In this study,Cancer Genome Atlas(TCGA)database was used to analyze the differentially expressed genes of LUSC and LUAD.Bioinformatics was used to identify these m RNA as potential molecular diagnostic markers to distinguish LUSC and LUAD.At the same time,the peripheral blood of 16 cases of lusc and 54 cases of luad were collected,the serum exosomes were isolated and identified,and the differential expression of m RNA could be used to distinguish LUSC and LUAD,and its relationship with clinical biological indicators was verified.The biological functions of these differentially expressed genes were analyzed through a variety of in vitro experiments in order to clarify the possible mechanism of their action in the occurrence and development of lung cancer and provide experimental basis for the follow-up study of the mechanism of exosomes in lung cancer.The main research contents and results are as follows:Part I Screening for differentially expressed genes in LUSC and LUAD by TCGA databaseObjective:The purpose of this part is to analyze the differentially expressed genes of LUSC and LUAD in the Cancer Genome Atlas(TCGA)database,in order to find potential molecular diagnostic markers to distinguish LUSC and LUAD.Methods:1.The differentially expressed genes of LUSC and LUAD were screened by TCGA database.2.GO analysis and KEGG pathway analysis of differentially expressed genes in LUSC and LUAD.3.According to the differentially expressed genes,the key genes were selected and their expression,clinical characteristics and prognosis of lung cancer patients were analyzed.Results:1.There were 1619 gene expression differences between LUSC and LUAD patients.Finally,17 differentially expressed genes(DEGs)with TPM >1000 were screened from lusc and luad groups.Among them,8 genes(S100A9,TP63,SFN,KRT17,KRT5,KRT6 a,HSPB1 and KAT19)were up-regulated in LUSC and down regulated in LUAD;9 genes(MT-ND6,CD74,HLA-DRB1,HLA-DRA,CEACAM6,SLC34A2,SFTPB,SFTPA1 and SFTPA2)were up regulated in LUAD and down regulated in LUSC.2.In LUAD,DEGs are mainly involved in a variety of biological processes,such as complement activation regulation,signal pattern recognition receptor activity,pattern recognition receptor signal pathway,negative regulation of epithelial to mesenchymal transformation;in LUSC,it is mainly involved in a variety of biological processes,such as cell division,mitotic mitosis,mitotic cell cycle G1/S transformation,mitotic sister chromatid separation,etc.3.The three most abundant signaling pathways in LUAD are complement and coagulation cascade,Staphylococcus aureus infection and cell adhesion molecules.However,the three most abundant signaling pathways in LUSC are cell cycle,p53 signaling pathway and tumor development.4.TP63 m RNA had no correlation with clinical stage,T stage,lymph node metastasis and distant metastasis of LUSC(P > 0.05);KRT5 m RNA had no correlation with clinical stage,T stage,lymph node metastasis and distant metastasis of LUSC(P > 0.05);CEACAM6 m RNA had no correlation with clinical stage,T stage,lymph node metastasis and distant metastasis of LUAD(P > 0.05);There was no correlation between SFTPB expression and clinical stage,T stage and distant metastasis of LUAD(P > 0.05),but there was a positive correlation between expression and lymph node metastasis(P <0.05).5.When TP63 was highly expressed,the survival rate of patients with LUAD decreased,but the difference was not statistically significant(P > 0.05);when TP63 was low,the survival rate of patients with LUSC decreased significantly(P < 0.01).When KRT5 was highly expressed,the survival rate of patients with LUAD decreased,but the difference was not statistically significant(P > 0.05);when KRT5 was low,the survival rate of patients with LUSC decreased significantly(P < 0.05).The survival rate of LUAD patients with low expression of CEACAM6 was lower,but the difference was not statistically significant(P > 0.05);the survival rate of LUSC patients with high expression of CEACAM6 was significantly lower(P < 0.01).The survival rate of LUAD patients with high expression of SFTPB decreased significantly(P < 0.01);the survival rate of LUSC patients with high expression of SFTPB decreased significantly(P <0.05).Summary:1.17 differentially expressed genes with TPM > 1000 were screened from LUSC and LUAD groups.Among them,8 of 17 DEGs were up-regulated in LUSC and down regulated in LUAD;9 of 17 DEGs were up regulated in LUAD and down regulated in LUSC.2.Go analysis and KEGG analysis of different genes were carried out by using bioinformatics method.The different genes in different histological types of lung cancer have different enrichment signal pathways.3.TP63 and KRT5 were up-regulated in LUSC,but down regulated in LUAD;CEACAM6 and SFTPB were up regulated in LUAD,but down regulated in LUSC.4.The expression of sftpb m RNA was positively correlated with lymph node metastasis in patients with LUAD,but not with other clinical features.The expression of CEACAM6 m RNA was not related to the clinical characteristics of patients with LUAD.TP63 m RNA and KRT5 m RNA were not related to the clinical characteristics of patients with LUSC.5.SFTPB can predict the prognosis of patients with LUSC and LUAD.TP63,KRT5 and CEACAM6 can predict the prognosis of patients with LUSC.Part Ⅱ Identification of TCGA screened differential genes in exosomes of LUSC and LUADObjective:The purpose of this study is to determine the expression of TP63,KRT5,CEACAM6 and SFTPB in exosomes and whether they can be used to identify LUSC and LUAD.Methods:Exosomes were extracted from the blood of 16 patients with squamous cell carcinoma of the lung and 54 patients with adenocarcinoma of the lung by ultrahigh speed centrifugation.The exosomes were analyzed and identified by transmission electron microscopy,Western blotting and NTA.The expression of TP63,KRT5,CEACAM6 and SFTPB m RNA in exosomes was detected,and the clinical diagnostic performance of squamous cell carcinoma and adenocarcinoma of the lung was evaluated by using the above differential genes alone or in combination.Results:1.The exosomes extracted from plasma were analyzed by transmission electron microscopy,Western blotting and NTA.2.Taking ACTB and SLC25 A as internal references,the expression levels of TP63 and KRT5 m RNA in exosomes of patients with lusc were significantly increased(P < 0.01),while the expression levels of CEACAM6 and SFTPB RNA in exosomes of patients with luad were significantly increased(P < 0.01,P < 0.05)3.The AUC of TP63 from exosomes was 0.682(95% CI: 0.526-0.837),the sensitivity and specificity were 74.1% and 62.5%,respectively.The AUC of KRT5 rom exosomes was 0.683(95% CI: 0.525-0.842),the sensitivity and specificity were 79.6% and 62.5%,respectively.The AUC of CEACAM6 from exosomes was 0.681(95% CI: 0.547-0.814),the sensitivity and specificity were 87.5% and 53.7%,respectively.The AUC of SFTPB from exosomes was 0.686(95% CI: 0.533-0.838),the sensitivity and specificity were 43.7% and 90.7%,respectively.The AUC of TP63,KRT5,CEACAM6 and SFTPB is 0.757(95% CI: 0.625-0.889),the sensitivity is 75.0%,the specificity is 72.2%,which is better than any of the above single genes.The AUC of TP63 from exosomes was 0.716(95% CI: 0.560-0.872),and the sensitivity and specificity were 61.1% and 75.0%,respectively.The AUC of KRT5 from exosomes was 0.715(95% CI: 0.586-0.844),the sensitivity and specificity were 81.3% and 66.7%,respectively.The AUC of CEACAM6 from exosomes was 0.730(95% CI: 0.592-0.869),the sensitivity and specificity were 81.3% and 66.7%,respectively.The AUC of SFTPB from exosomes was 0.701(95% CI: 0.548-0.855),the sensitivity and specificity were 50.0% and 87.0%,respectively.The AUC of TP63,KRT5,CEACAM6 and SFTPB is 0.822(95% CI: 0.699-0.945),the sensitivity is 62.5%,the specificity is 88.89%,which is better than any of the above single genes.Summary:1.TP63 and KRT5 from exosomes increased in lusc and decreased in luad in NSCLC patients.2.The expression of CEACAM6 and SFTPB from exosomes in sera of patients with NSCLC increased in luad and decreased in LUSC.3.AUC of NSCLC was 0.682,0.683,0.681 and 0.686 based on ACTB.Based on ACTB as internal reference,AUC,sensitivity and specificity of TP63,KRT5,CEACAM6 and SFTPB are superior to those of any single marker.4.AUC of NSCLC was 0.716,0.715,0.730 and 0.701 based on SLC25 A.Based on SLC25 A as internal reference,AUC,sensitivity and specificity of TP63,KRT5,CEACAM6 and SFTPB are superior to those of any single marker.Part Ⅲ Expression and role of TP63,KRT5,CEACAM6 and SFTPB in NSCLC cellsObjective:To investigate the expression of TP63,KRT5,CEACAM6 and SFTPB in NSCLC cells and their effects on the proliferation,migration and apotosis of NSCLC cells.Methods:q RT-PCR was used to detect the expression of TP63,KRT5,CEACAM6 and SFTPB in NSCLC cells.The effects of overexpression or knockdown of TP63,KRT5,CEACAM6 and SFTPB on the proliferation,migration invasion and apotosis of NSCLC cells were examined by CCK-8,scratch healing,transwell experiments and flow cytometry.Results:1.The results of q RT-PCR showed that the expression of TP63 and KRT5 m RNA in H1299 cells was higher than that in H975 cells,and the expression of CEACAM6 and SFTPB m RNA in 975 cells was higher than that in H1299 cells.2.CCK-8 assay showed that the proliferation of H1299 cells was decreased significantly after treatment of TP63 overexpression(P < 0.05),while the proliferation of H1299 cells was increased significantly after treatment of KRT5 overexpression(P < 0.05);the proliferation of H1975 cells was increased significantly after treatment of CEACAM6 overexpression(P <0.05),while the proliferation of H1975 cells was decreased significantly after treatment of SFTPB overexpression(P < 0.05);the proliferation of H1975 cells was increased significantly after treatment of TP63-si RNA(P < 0.05),while the proliferation of H1975 cells was decreased significantly after treatment of KRT5-si RNA(P < 0.05);the proliferation of H1299 cells was decreased significantly after treatment of CEACAM6-si RNA(P < 0.05),while the proliferation of H1299 cells was increased significantly after treatment of SFTPB-si RNA(P < 0.05).3.Scratch-healing experiments showed that the migration of H1299 cells was decreased significantly after treatment of TP63 overexpression(P < 0.05),while the migration of H1299 cells was increased significantly after treatment of KRT5 overexpression(P < 0.05);the migration of H1975 cells was increased after treatment of CEACAM6 overexpression in some extent but the difference was not statistically significant(P > 0.05),while the migration of H1975 cells was increased significantly after treatment of SFTPB overexpression;the migration of H1975 cells was increased significantly after treatment of TP63-si RNA(P < 0.05),while the migration of H1975 cells was decreased significantly after treatment of KRT5-si RNA(P < 0.05);the migration of H1299 cells was decreased significantly after treatment of CEACAM6-si RNA(P < 0.05),while the migration of H1299 cells was increased significantly after treatment of SFTPB-si RNA(P < 0.05).4.Flow cytometry showed that overexpression of TP63 could promote the apoptosis of H1299 cells(P < 0.05),while overexpression of KRT5 could inhibit the apoptosis of H1299 cells(P < 0.05);overexpression of CEACAM6 could inhibit the apoptosis of H1975 cells(P < 0.05),overexpression of SFTPB could promote the apoptosis of H1975 cells(P < 0.05);knockdown of TP63 could inhibit the apoptosis of H1975 cells(P < 0.05);knockdown of KRT5,CEACAM6 and SFTPB could promote the apoptosis of H1299 cells(P< 0.05).Summary:1.TP63 and KRT5 m RNA were highly expressed in lung squamous cell carcinoma cell line H1975,but low in lung adenocarcinoma cell line H1299.2.The expression of CEACAM6 and SFTPB m RNA was high in lung adenocarcinoma cell line H1299,but low in lung squamous cell carcinoma cell line H1975.3.Overexpression of TP63 in H1299 cells can inhibit the proliferation and migration of H1299 cells and promote the apoptosis of H1299 cells;overexpression of KRT5 in H1299 cells can promote the proliferation and migration of H1299 cells and reduce the apoptosis of H1299 cells;knocking down TP63 in H1975 cells can promote the proliferation and migration of H1975 cells and reduce the apoptosis of H1975 cells;knocking down KRT5 in H1975 cells can promote the proliferation and migration of H1975 cells and promote the apoptosis of H1975 cells.4.Overexpression of CEACAM6 in H1975 cells can promote the proliferation of H1975 cells and reduce the apoptosis of H1975 cells;overexpression of SFTPB in H1975 cells can inhibit the proliferation and migration of H1975 cells and promote the apoptosis of H1975 cells;knockdown of CEACAM6 in H1299 cells can inhibit the proliferation and migration of H1299 cells and reduce the apoptosis of H1299 cells;knockdown of SFTPB in H1299 cells can promote the proliferation and migration of H1299 cells and reduce the apoptosis of H1299 cells.Conclusions:1.TP63,KRT5,CEACAM6 and SFTPB m RNA from exosomes may be potential biomarkers to distinguish LUAD and LUSC.The combination of multiple biomarkers can improve the specificity and sensitivity of the diagnosis of different lung cancer subtypes.2.TP63,KRT5,CEACAM6 and SFTPB affect the proliferation,migration and apoptosis of lung cancer cells. |
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