Bioinformatics Analysis Of Genes Related To Lung Adenocarcinoma Metastasis And Its Role And Molecular In Lung Adenocarcinoma

Background and objective:Lung cancer is the most common malignant tumor with the highest morbidity and mortality worldwide,and is one of the leading causes of cancer-related death.According to the National Cancer Center of China,the incidence of lung cancer accounts for the first place in the nation's malignant tumors.The number of new cases has risen to 800,000 cases per year,ranking first in the incidence of male cancer,and second only to breast cancer in the incidence of female cancer.Take the second place.Lung cancer mortality is also the leading cause of malignant tumor-related deaths nationwide,with an annual death toll of about 700,000.It ranks first in both male and female cancer-related deaths.The pathogenesis involves multiple steps such as proto-oncogene activation,tumor suppressor gene inactivation,induction of angiogenesis,growth inhibition escape,regulation of energy metabolism,self-feedback secretion loop activation,immune escape,inhibition of apoptosis,etc.,resulting in tumor cell growth.Loss of control ultimately leads to dysregulation of cellular physiological functions such as proliferation,differentiation,signal transmission,exercise,and apoptosis.Early lung cancer can achieve better therapeutic effect through surgical treatment,but most lung cancer has local invasion or distant metastasis at the early stage of diagnosis,and cancer-related death is mostly caused by lung cancer invasion and distant metastasis.These treatments have effectively improved the survival and quality of life of patients with advanced metastatic lung cancer,but due to secondary drug resistance,the overall prognosis of patients with metastatic lung cancer is poor,and the 2-year survival rate is still very low.With the rapid development of gene chip technology and high-throughput sequencing technology,bioinformatics analysis is widely used in the field of tumor gene function research,providing massive bioinformatics data for tumor science research,making cancer research into multi-dimensional comprehensive analysis.the height of.The integrated analysis of cancer-related biological information in biological databases has become an effective means to study oncogenes.The GEO database is currently the largest and most comprehensive public database for storing gene chips and high-throughput sequencing expression profiles.The Cancer Genome Atlas(TCGA)database contains multiple cancer genomics data,including epigenetic data,transcriptome data,gene mutation data,proteomic data,and clinical data for each cancer.The development of related mechanisms and the screening of key genes provide a wealth of resources.The open database resources provide cancer researchers with a large number of cancer genetic and epigenetic data,reasonably and effectively integrate these public data,and quickly discover cancer biomarkers and therapeutic targets for cancer diagnosis and individualized precision treatment.And provide a basis for prevention.Based on the GEO database,this study screened the gene chip sample set related to lung adenocarcinoma metastasis,and obtained the differentially expressed gene set of high metastatic potential of lung adenocarcinoma by R language analysis,and analyzed the GO function enrichment for differentially expressed genes.Pathway analysis,differential expression gene analysis of the sample set to obtain co-expressed differential genes,survival analysis on the GEPIA data platform,online clinical analysis platform such as Oncomine,Kaplan-Meier Plotter and other clinical information on lung adenocarcinoma in TCGA and other databases Co-expressed genes were validated,and the gene SLC44A1,which is differentially expressed in lung adenocarcinoma cells and adjacent tissues and closely related to the overall survival time of patients,was selected as the key gene for lung adenocarcinoma metastasis.We first analyzed the association with clinicopathological features in paired lung adenocarcinoma and adjacent tissues.The expression of SLC44A1 protein in lung adenocarcinoma tissues was verified by immunohistochemistry and its correlation with prognosis was analyzed.Subsequently,we studied the effects of SLC44A1 on the proliferation,invasion,migration,apoptosis,self-renewal ability,transplantation and tumor formation of lung adenocarcinoma Calu3 and H322 in vivo and in vitro experiments,and finally influenced the SLC44A1 from immune infiltration.The mechanism of lung adenocarcinoma metastasis was discussed.Part 1 Bioinformatics analysis and screening of genes related to lung adenocarcinoma metastasis Methods 1.Search in the GEO Data Sets with "lung adenocarcinoma" and "metastasis" as keywords to select a sample set that is consistent with lung adenocarcinoma metastasis.2.Download and save the sample set CEL data file and the corresponding platform probe file respectively.The chip software is preprocessed using R software.3.The metastatic group was used as the experimental group and the non-transfer group was used as the control group.The differentially expressed genes were analyzed for the two sample sets using the limma package in the R software,and significant differential genes were screened by using |log FC|>1 and adj.p<0.05 as inclusion criteria.4.Co-expression analysis of differential genes in the sample set to obtain key genes related to lung adenocarcinoma metastasis.5.Prognostic verification of significant differential genes on the GEPIA online platform,and select genes with high correlation with the prognosis of lung adenocarcinoma as key genes.Results1.Two gene chip sample data sets GSE76194 and GSE42407 related to lung adenocarcinoma metastasis were screened,and the gene chip platform files of the above two sample sets were GPL570.2.The sample set GSE76194 lung adenocarcinoma contains 8 samples,and the sample set GSE42407 contains 6 samples.The matrix data after pretreatment has good standardization effect and can be analyzed later.3.After obtaining differentially expressed genes,significant differential genes were screened by using |log FC|>1 and adj.p<0.05 as the inclusion criteria.There were 199 significant differential genes,107 up-regulated genes and 92 down-regulated genes in GSE76194.There are 3278 significant differential genes in GSE42407,2601 up-regulated genes and 677 down-regulated genes.4.Co-expression analysis of the two groups of differential genes revealed that there were 22 co-expressed genes in the two sample sets,including 18 up-regulated genes and 4 down-regulated genes.5.GO function enrichment analysis and Pathway analysis of co-expressed genes showed that their biological functions enriched cancer-related pathways.6.Through the GEPIA online platform verification,SLC44A1 was significantly associated with the prognosis of patients,and the survival rate of patients with high expression was significantly lower than that of the low expression group.Summary 1.Differentially expressed genes of lung adenocarcinoma metastasis-related gene chips GSE76194 and GSE76194 were analyzed,and 18 up-regulated genes and 4 down-regulated genes were screened after co-expression analysis of differential samples of two sample sets.2.Through online survival verification,it is speculated that SLC44A1 plays an important role in the occurrence,invasion and metastasis of lung adenocarcinoma.Part 2 Expression and clinical significance of SLC44A1 in lung adenocarcinoma Methods 1.Analysis of the expression levels of SLC44A1 in different cancers in the Oncomine and TIMER databases.2.Download RNA-seq data of lung adenocarcinoma in TCGA,and analyze the expression of SLC44A1 in clinical samples of lung adenocarcinoma.The clinical information data were matched to analyze the survival correlation of SLC44A1 in clinical samples of lung adenocarcinoma.3.The expression of SLC44A1 m RNA in paired lung adenocarcinoma tissues and adjacent normal tissues was detected by Real Time-PCR,and its correlation with clinicopathological parameters was analyzed.4.The expression of SLC44A1 protein in lung adenocarcinoma tissues and adjacent normal tissues was detected by immunohistochemistry,and its correlation with clinicopathological parameters was analyzed.5.To analyze the correlation between the expression level of SLC44A1 and the survival prognosis of patients.Results 1.Oncomine and TIMER database results show that SLC44A1 is highly expressed in breast cancer,cervical cancer,head and neck cancer,lung cancer,lymphoma,pancreatic cancer,etc.;low in bladder cancer,breast cancer,colorectal cancer,kidney cancer,liver cancer expression.2.TCGA database found that SLC44A1 expression in lung adenocarcinoma tissue was higher than normal tissue,metastatic lung adenocarcinoma(M1)expression was higher than non-metastatic lung adenocarcinoma(M0),the difference was statistically significant.3.The expression of SLC44A1 m RNA in 68 matched lung adenocarcinoma tissues was significantly higher than that in adjacent normal tissues(p<0.05),and its expression level in cancer tissues was significantly correlated with lymph node metastasis,tumor stage and survival status.<0.05).4.The expression level of SLC44A1 protein in lung adenocarcinoma tissues was significantly higher than that in adjacent normal tissues(p<0.05),and its expression in cancer tissues was significantly correlated with lymph node metastasis,tumor stage and survival status(p<0.05).5.The expression of SLC44A1 is related to the survival of patients,and the prognosis of patients with high expression is worse.Summary 1.SLC44A1 is highly expressed in various cancers including lung adenocarcinoma.2.SLC44A1 expression in lung adenocarcinoma tissue is higher than adjacent normal tissues,and is related to the patient's primary tumor range,lymph node metastasis,tumor stage.Patients with high expression have a worse prognosis.It is suggested that SLC44A1 may be involved in the malignant progression of lung adenocarcinoma.Part 3 Effect of SLC44A1 on biological behavior of lung adenocarcinoma cells Methods 1.Real Time-PCR was used to detect the expression of SLC44A1 m RNA in three human lung adenocarcinoma cell lines and human normal lung bronchial epithelial cell lines.2.SLC44A1 stably knockdown lung adenocarcinoma cell line was constructed,and the knockdown efficiency of SLC44A1 was verified by Western Blot and Real time-PCR.3.Plate cloning assay and cell proliferation assay(CCK8)were used to detect the effect of knockdown of lung adenocarcinoma SLC44A1 on cell proliferation.4.Scratch and Transwell experiments were used to detect the effect of SLC44A1 knockdown on the invasion and migration of lung adenocarcinoma cells.5.The effect of SLC44A1 knockdown on apoptosis was detected by flow cytometry.6.Ultra-low adhesion into a ball culture experiment to test the effect of SLC44A1 knockdown on the self-renewal ability of lung adenocarcinoma.7.Construct a nude mouse lung adenocarcinoma xenograft model,observe the tumor growth,and detect the expression of SLC44A1 and proliferation and invasion related indicators in the tumor site.Results 1.The expression of SLC44A1 in lung adenocarcinoma cell lines A549,Calu-3 and H322 was significantly higher than that in human lung bronchial epithelial cell line BEAS-2B.2.Select lung cancer cell lines Calu-3 and H322 with relatively high expression of SLC44A1 to construct a cell line with stable knockdown.3.Knockdown of SLC44A1 gene can inhibit the proliferation,invasion and migration of Calu-3 and H322,and promote the apoptosis of Calu-3 and H322 cells,but has no significant effect on the self-renewal ability of lung adenocarcinoma cells.4.In the tumor-forming experiment of nude mice,knockdown of SLC44A1 gene can inhibit the tumorigenic ability of nude mice,and the expression of SLC44A1 and proliferation-invasive indicators ki-67 and MMP9 are down-regulated.1.In vitro cell experiments,SLC44A1 knockdown inhibits proliferation,invasion,migration,and promotes apoptosis of lung adenocarcinoma cells.2.In vivo animal experiments,SLC44A1 knockdown can inhibit the tumorigenic ability of nude mice,suggesting that SLC44A1 plays an important role in the malignant progression of lung adenocarcinoma.Summary 1.In vitro cell experiments,SLC44A1 knockdown inhibits proliferation,invasion,migration,and promotes apoptosis of lung adenocarcinoma cells.2.In vivo animal experiments,SLC44A1 knockdown can inhibit the tumorigenic ability of nude mice,suggesting that SLC44A1 plays an important role in the malignant progression of lung adenocarcinoma.Part 4 Mechanism of SLC44A1 Affecting Invasion and Migration of Lung Adenocarcinoma Methods 1.Multi-factor detection technique and RT-PCR analysis of the secretion level of chemokines in Calu-3 and H322 cells after 48 min of SLC44A1 knockdown,and find the most significant difference.2.The expression of differential chemokines was verified by ELISA.3.Correlation analysis of SLC44A1 and related immune factors on the GEPIA platform.4.Real Time-PCR was used to detect the expression of SLC44A1,CD68,CD163,CCL2 and m RNA in human lung adenocarcinoma,and the mechanism of SLC44A1 affecting lung adenocarcinoma metastasis was discussed.Results 1.Multi-factor detection,multi-factor RT-PCR and ELISA showed that the level of CCL2 secreted by H322 cells decreased after SLC44A1 knockdown.2.Database analysis showed that SLC44A1 was positively correlated with CCL2,CD14,CD68,CD163,IL-10 and IL-6.3.SLC44A1 was positively correlated with CD68,CD163 and CCL2 in lung adenocarcinoma tissues,suggesting that it may play a tumor-promoting role by regulating the polarization of TAMs to M2.Summary After SLC44A1 knockdown,the level of CCL2 secreted by the supernatant of H322 cells decreased.The database and clinical samples confirmed that SLC44A1 was positively correlated with CD68,CD163,CD14,IL-10 and IL-6,and positively correlated with CCL-2 of chemotactic TAMs.It is speculated that it may play a role in promoting tumorigenesis by regulating the polarization of M2 TAMs.Conclusion 1.Through bioinformatic analysis of genes related to lung adenocarcinoma metastasis,22 genes differentially expressed in lung adenocarcinoma were screened.Through survival prognosis,it is speculated that SLC44A1 is a key gene in lung adenocarcinoma metastasis.2,verified by multiple database platforms,SLC44A1 is highly expressed in a variety of cancers including lung adenocarcinoma.The overall survival rate and disease-free survival rate of patients with high expression of SLC44A1 in human lung adenocarcinoma were lower than those with low expression,suggesting that it is an indicator of poor prognosis.3.SLC44A1 knockdown inhibits the proliferation,invasion and migration of lung adenocarcinoma cells,promotes apoptosis and inhibits tumorigenic ability.It is speculated that this gene plays an important role in the malignant progression of lung adenocarcinoma.4.After SLC44A1 knocked down,the level of CCL2 secreted by H322 cells decreased.Immunoinfiltration analysis showed that SLC44A1 was positively correlated with CD68 and CD163,and positively correlated with CCL-2 of chemotactic TAMs.It is speculated that it may play a tumor-promoting role by regulating the polarization of M2 TAMs.