Screening And Functional Studies Of Key Genes Related To Lung Adenocarcinoma Metastasis

Epidemiological data show that lung cancer is one of the malignant tumors with the highest incidence mortality in my country,and the 5-year survival rate is only 20%.Non-small cell lung cancer is the most common pathological type of lung cancer,and lung adenocarcinoma is the main pathological subtype.Malignant invasion and metastasis of lung adenocarcinoma are the main causes of poor prognosis.The application of small-molecule tyrosine kinase inhibitors(TKIs)has changed the therapeutic effect of lung adenocarcinoma,but most patients will eventually develop resistance.Immune checkpoint inhibitor(ICS)therapy has achieved revolutionary efficacy,but the vast majority of patients are insensitive to immunotherapy.So,it is very critical to explore the metastasis mechanism of lung adenocarcinoma and find the key genes that affect the metastasis of lung adenocarcinoma so as to control its invasion and metastasis,which will improve the prognosis of lung adenocarcinoma patients.Current studies have shown that the metastasis of lung adenocarcinoma is a complex process of multiple factors and multiple genes acting together,and the specific mechanism is still unclear.In recent years,omics research has flourished,and the formation of a large amount of data such as single-cell sequencing,exome sequencing,and methylation sequencing has provided data support for a deep understanding of the mechanism of tumor occurrence and development,and has played an important role in the early screening,diagnosis,and treatment of tumors.It plays an important role in monitoring recurrence and other processes.By using advanced bioinformatics technology to conduct high-precision and systematic analysis and mining of single-cell transcriptome sequencing data and bulk sequencing data,it is possible to obtain the gene expression differences at the cell level of common cancers and discover what may be lost in traditional bulk tissue sequencing.Significant genes,molecular biomarkers for new post-diagnosis prognosis.Therefore,analyzing and mining the single-cell transcriptome data and bulk sequencing data of lung adenocarcinoma is of great significance for elucidating the metastasis mechanism of lung adenocarcinoma and obtaining biomarkers of lung adenocarcinoma metastasis and prognosis.In this study,the single-cell transcriptome data of lung adenocarcinoma were downloaded from the Gene Expression Omnibus(GEO)database,and the transcriptome sequencing data of lung adenocarcinoma and adjacent paracancerous tissues were downloaded from the Tumor Genome Dataset(The Cancer Genome Atlas,TCGA).First,the single-cell data of lung adenocarcinoma were clustered and grouped,and the cancer epithelial cells were identified.Through the pseudo-chronological analysis of the cancer epithelial cells of the primary tumor and distant metastases,the gene set related to the metastasis of lung adenocarcinoma was identified.Then,we combined the lung adenocarcinoma bulk sequencing data and clinical information of the TCGA database,and performed LASSO regression analysis on the metastasis-related gene sets identified by the single-cell transcriptome data to screen out key genes related to prognosis.Collect clinical samples,verify the expression of these key genes in lung adenocarcinoma and adjacent paracancerous tissues,and analyze the correlation of gene expression with different clinical features of lung adenocarcinoma.Then,through in vitro cell experiments,the selected key genes were overexpressed and silenced,and their effects on the biological functions of lung adenocarcinoma cell lines,such as proliferation,migration,invasion,and clone,were studied both positively and negatively.This study will help to develop new diagnostic and prognostic biomarkers for lung adenocarcinoma,and provide a new direction for finding new therapeutic targets for lung adenocarcinoma.Part one Screening key genes related to lung adenocarcinoma metastasis based on single-cell combined bulk sequencing dataObjective: To systematically analyze single-cell data of cancer epithelial cells from primary and metastatic lung adenocarcinoma in order to obtain and analyze a data set of metastasis-related genes.Methods:1.Download the GSE131907 microarray data from the GEO database,and we select 4 cases of primary and 4 cases of lung adenocarcinoma with pleural metastases for single-cell analysis.2.Pseudochronological analysis of primary and pleural metastatic cancerous epithelial cells identifies post-metastasis-associated gene sets.Cox regression analysis was performed on differential genes to identify prognostic-related genes.A prognostic model was constructed,and finally the most critical prognostic genes were screened.Results:1.By analyzing the single-cell transcriptome data of primary and metastatic lung adenocarcinoma,126 metastasis-related differential genes were obtained,which were defined as lung adenocarcinoma metastasis-associated genes(MAGs).2.Validated MAGs in the TCGA-LUAD dataset,obtained three metastasis-associated molecular subtypes,and constructed a risk model.In addition,P4HA1 was found to be the core gene,which may play an important role in the occurrence and development of lung adenocarcinoma.Conclusinon: P4HA1 is the core gene,which may play an important role in the occurrence and development of lung adenocarcinoma.Part two Role of P4HA1 in pan-carcinoma and lung adenocarcinomaObjective: To conduct bioinformatics analysis of P4HA1 in pan-cancer and lung adenocarcinoma.Methods:1.Download P4HA1-related expression profile data and clinical information,as well as immune cell infiltration data from the online database.2.Use R language to conduct bioinformatics analysis on the role of P4HA1 in pan-cancer.3.Comprehensive bioinformatics analysis of P4HA1 in lung adenocarcinoma.Results:1.P4HA1 is highly expressed in 26 kinds of tumors,including lung adenocarcinoma.The level of promoter methylated P4HA1 was inversely correlated with P4HA1 expression.2.The results of Kaplan-Meier analysis showed that high expression of P4HA1 was associated with poorer survival of various tumors.GSEA pathway enrichment analysis showed that: P4HA1 is involved in pan-cancer immune regulation related pathways,and the high expression of P4HA1 is related to the suppression of tumor immune microenvironment.3.P4HA1 was significantly overexpressed in lung adenocarcinoma and correlated with poor prognosis.P4HA1 expression positively correlated with immunosuppressive cell expression.Conclusions: P4HA1 is highly expressed in a variety of tumor tissues in pan-cancer and is associated with poor prognosis,including lung adenocarcinoma.High expression of P4HA1 is associated with tumor immunosuppressive microenvironment.Part three Expression verification of P4HA1 in lung adenocarcinoma and its correlation with clinicopathological featuresObjective: To verify the expression of P4HA1 in lung adenocarcinoma and adjacent paracancerous tissues,and to study the correlation between the expression of P4HA1 and different clinicopathological features of lung adenocarcinoma.Methods:1.Collect surgical specimens from patients with lung adenocarcinoma and register the clinical information of the patients.2.Explore the expression of P4HA1 in lung adenocarcinoma tissues and adjacent tissues by q RT-PCR.3.Western Blot(WB)detects the expression level of the P4HA1 protein between lung adenocarcinoma tissue and adjacent tissue.4.Immunohistochemical method was used to detect the expression of P4HA1 in lung adenocarcinoma and adjacent paracancerous tissues.5.To analyze the correlation between P4HA1 expression and different clinicopathological features of lung adenocarcinoma patients.Results:1.The results of q RT-PCR showed that the relative expression level of P4HA1 in lung adenocarcinoma tissue was significantly higher than that in paracancerous tissue(P<0.01).2.The results of western blot detection showed that the expression of P4HA1 protein in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues(P<0.05).3.The results of immunohistochemical detection showed that the expression of P4HA1 in lung adenocarcinoma was significantly higher than that in adjacent cancerous tissues(P<0.05).4.The expression of P4HA1 was correlated with lymph node metastasis and TNM stage of patients(P<0.05).Conclusions: P4HA1 expression is significantly higher in lung adenocarcinoma than in adjacent paracancerous tissues,and is closely related to whether patients had lymph node metastasis and TNM stage.Part four Effect of P4HA1 on the malignant behavior of lung adenocarcinoma cell linesObjective: To observe the effect of P4HA1 gene on the biological functions of lung adenocarcinoma cell line proliferation,migration,invasion and cloning by overexpressing and knocking down P4HA1 gene in lung adenocarcinoma cell line.Methods: P4HA1 was knocked down and overexpressed in H-1299 and A549 lung adenocarcinoma cell lines,and the effects of P4HA1 on lung adenocarcinoma cells were studied by cell proliferation assay(CCK-8method),cell scratch assay,Transwell assay and plate cloning assay.Effects on proliferation,migration,invasion and cloning.Results:1.The results of q RT-PCR showed that in the H1299 lung adenocarcinoma cell line,the expression of P4HA1 m RNA was significantly decreased in the si-P4HA1 group knocking down P4HA1(P<0.05).In the A549 lung cancer cell line,the expression of P4HA1 m RNA was significantly increased in the pc DNA3.1/P4HA1 group overexpressing P4HA1(P<0.05).2.The results of CCK-8 showed that in the H1299 lung adenocarcinoma cell line,the si-P4HA1 cell group with P4HA1 knocked down,the cell proliferation ability was weakened at 48 h and 72 h(P<0.05).In the A549 lung cancer cell line,the proliferation ability of the pc DNA3.1/P4HA1 cell group overexpressing P4HA1 was significantly enhanced at 24 h,48 h,and 72h(P<0.05).3.The results of the cell scratch test showed that in the H1299 lung adenocarcinoma cell line,the migration distance of the P4HA1-knockdown si-P4HA1 group was significantly reduced(P<0.05).In the A549 cell line,the migration distance of the pc DNA3.1/P4HA1 group overexpressing P4HA1 was significantly increased(P<0.05).4.The results of Transwell experiments showed that in the H1299 cell line,the number of invasive cells in the P4HA1-knockdown si-P4HA1 group was significantly reduced(P<0.05).In the A549 cell line,the number of invasive cells in the pc DNA3.1/P4HA1 group overexpressing P4HA1 was significantly increased(P<0.05).5.The results of the plate cloning experiment showed that in the H1299 cell line,the colony formation rate of the si-P4HA1 group knocking down P4HA1 was significantly reduced(P<0.05).In the A549 cell line,the colony formation rate of the pc DNA3.1/P4HA1 group overexpressing P4HA1 was significantly increased(P<0.05).Conclusion: P4HA1 promotes the proliferation,migration,invasion and formation of clone of lung adenocarcinoma cells.