The Role Of Methylation-associated Genes CRYAB And SLC39A11 In The Development Of Lung Adenocarcinoma

Background and objectives: lung adenocarcinoma(LUAD)is one of the most common lung cancer,accounting for about 40% of all lung cancer cases.Due to the lack of accurate early diagnosis standard molecular markers,at present,the diagnosis of LUAD is usually in the middle and late stage.If it is diagnosed in the early stage,the five-year survival rate of patients is 54%,but if it is diagnosed in the late stage,the five-year survival rate of patients is less than 20%.There are also many studies on the molecular mechanism of LUAD,and the use of multiomics research,including transcriptome sequencing to screen differential gene expression and methylation chip sequencing to identify differential methylation sites,can take into account a variety of factors and is more in line with the complexity of tumor molecular changes.What’s more,tumor typing can be realized.At present,such studies are limited in the small number of cases,so the results can not be popularized in clinical application.In addition,the occurrence and development of LUAD are closely related to the tumor microenvironment(TME).The TME the internal environment in which tumor cells produce and live,is characterized by hypoxia,chronic inflammation and immunosuppression.Immune cell infiltration is an important part of the TME.Immune cells and immune response play a very important role in the occurrence and development of LUAD.Therefore,the purpose of this study is to jointly analyze and find out the genes related to differential methylation sites related to prognosis based on the data of TCGA(the Cancer Genome Atlas)and GEO database,select specific genes for biological experimental verification,so as to clarify the efficacy of genes,provide new potential markers for early clinical diagnosis of LUAD and provide new ideas for the treatment of LUAD.Methods: The data of GSE32863 and GSE75037 were combined and standardized.The deferentially expressed genes were calculated by R limma package,and GO,KEGG were analyzed;The deferential expressed genes were further analyzed by LASSO and SVM regression analysis,and the genes of two regression analysis were combined and identified as diagnostic genes.The prediction efficiency of diagnostic genes was analyzed by AUC curve.CIBERSORT analysis was used to define the cell components of complex tissues based on gene expression,and the correlation between deferentially expressed genes and tissue infiltrating immune cells was analyzed.The differential expression of TCGA gene set and methylation sites ofLUAD were analyzed.Then,the relationship between these molecular subtypes and clinical characteristics was analyzed.The representative genes and methylation sites of LUAD subtypes were identified and analyzed by GO and KEGG.The molecular typing results of LUAD were externally verified using the data set GSE32863.Based on molecular typing,specific genes were selected to verify in the downstream phenotype experiment and further mechanism study.Results: 1.After integrating the data of GSE32863 and GSE75037 data sets,the limma package analysis found that there were 384 differential genes between the control group and the experimental group,of which 91 were up-regulated and 293 were down regulated.After the intersection of LASSO and SVM regression,seven genes were finally identified as diagnostic genes: LOC401286,FMO2,XLKD1,RHOJ,SCARA5,HSPB2 and SERINC2.The areas under the curve were 0.99,1.00,0.99,1.00,0.99,0.99 and 0.98 respectively.2.CIBERSORT analysis showed that Na ? ve B cells,memory B cells,plasma cells,Na ? ve CD4 T cells,TFH cells,Treg cells,γδ T cells,monocytes,M0 macrophages,M1 macrophages,tissue resident mast cells,activated mast cells and neutrophils are different between normal and tumor tissues,and these immune cells are related to the above diagnostic genes.LOC401286,FMO2,XLKD1,RHOJ,SCARA5 and HSBPS were a positive correlation between monocytes and resting mast cells,and a negative correlation with regulatory T cells and macrophages.3.Based on the comprehensive analysis of gene methylation and transcriptome data,LUAD were divided into two subtypes.The prognosis of subtype 1 was significantly worse than that of subtype 2.There were significant differences between the two subtypes in survival status,age,gender,tumor stage,N stage and clinical stage.The pivotal genes found in the pathway analysis related to cell cycle and immune system including NCAPG,CCNB1,DLGAP5,HLA-DQA1,HLA-DPA1,HLA-DPB1,SFTP,SCGBA1 A and SFTPD.These genes are related to cell cycle regulation,T cell recruitment,anti-pd1 treatment and anti-inflammatory process.The results of molecular typing were also verified in the data set GSE32863.3.Survival analysis showed that the low expression of CRYAB was significantly correlated with the poor prognosis of LUAD,and the high expression of SLC39A11 was significantly correlated with the poor prognosis of lung adenocarcinoma.4.Phenotypic experiment results: cck-8 experiment showed that knockout of CRYAB promoted the proliferation of tumor cells,and knockout of SLC39A11 inhibited the proliferation of tumor cells.Plate cloning experiment showed that knockout of CRYAB promoted the clonal proliferation of tumor cells,and knockout of SLC39A11 inhibited the clonal proliferation of tumor cells.The results of cell cycle and apoptosis experiments showed that knockout of CRYAB promoted tumor cells to enter the cell division stage and inhibited tumor cell apoptosis,while knockout of SLC39A11 reduced tumor cells in the division stage and promoted tumor cell apoptosis.Cell migration experiments showed that knockout of CRYAB promoted tumor cell migration,and knockout of s SLC39A11 inhibited tumor cell migration.Conclusions: 1 LOC401286,FMO2,XLKD1,RHOJ,SCARA5,HSPB2 and SERINC2 can be used as early diagnostic genes of LUAD.LOC401286 is the gene related to lung adenocarcinoma found for the first time,and further clinical validation research is still needed.2.Combining gene expression and methylation results can classify LUADs into two types.3.The expression of CRYAB and slc39a11 is associated with poor prognosis and methylation in lung adenocarcinoma.They can be used as a prognostic index of lung adenocarcinoma and is expected to become a therapeutic target of lung adenocarcinoma.