Analysis Of LncRNA Expression And Establishment Of Prognosis Signature Of Bladder Cancer Based On TCGA Database

Objective:To investigate the role of long non-coding RNA(lnccRNA)in the prognosis of patients with bladder cancer(BC)and to construct a prognostic signature of lncRNA.To explore the differences in molecular subtypes and immune cell infiltration among patients at different risk groups based on the prognostic signature of lncRNA.Methods:1.The RNA data and clinical data of all bladder cancer patients were downloaded from the Cancer Genome Atlas website(TCGA),and the difference analysis of lncRNA and mRNA was conducted to obtain the differentially expressed lncRNA and mRNA in cancer tissues and paracancerous tissues,respectively.2.All samples were screened and only those with complete clinical data were retained,and then the samples were randomly divided into the train group and the validation group.In the train group,univariate and multivariate Cox regression analysis was used to identify the lncRNAs with greater prognostic impact from the differentially expressed lncRNAs to construct the prognostic model,which was verified in the validation group and two GSE datasets.3.Through correlation analysis,the mRNA associated with lncRNA in the prognostic model(correlation coefficient>0.6)was found out,and the related mRNAs were analyzed by Protein-Protein interaction network analysis and GO/KEGG/GSEA pathway enrichment analysis.4.The established lncRNA prognostic signature was used to calculate the risk scores of all samples and all samples were divided into high-risk and low-risk groups.The Cibersort algorithm was used to calculate the proportion of 22 immune cell types in each sample.The differences in the abundance and types of immune cell infiltration were analyzed in the high and low risk groups.5.Combined with the existing molecular subtyping data of bladder cancer,the differences in molecular subtype in the high and low risk groups were analyzed.6.Fluorescence quantitative PCR was used to measure the relative expression of IncRNAs in the prognostic model of IncRNAs in clinical samples and four bladder cancer cell lines(T24、RT4、UMUC3 and SV-HUC-1).LncRNA was knocked down using shRNA,and invasion and migration of bladder cancer cells were observed.Results:1.614 differentially expressed lncRNAs and 4384 differentially express-ed mRNAs were found from the TCGA-BLCA cohort.2.Six IncRNAs(SNHG12,MAFG-AS1,ASMTL-AS1,LINC02321,LINC01322 and LINC00922)were identified from the differentially expressed IncRNAs to construct the prognostic signature,named SMALLL.Survival analysis showed that the prognosis signature of SMALLL had significant prognostic power for the overall survival rate of bladder cancer patients in each group(train group,validation group,GSE31684 and GSE32894).3.Pathway enrichment analysis showed that genes co-expressed with SMALLL signature were related to extracellular matrix network.4.Analysis of immune cell infiltration showed that the abundance of naive B cells,regulatory T cells(Tregs),macrophages M0,eosinophils,resting memory CD4 T cells and resting NK cells were significantly different in the high and low risk groups.5.In general,molecular subtype analysis showed that most of the high-risk group was Basel type,while the low-risk group was Luminal type.6.Fluorescence quantitative RT-PCR analysis confirmed the differential expression of lncRNA in SMALLL characteristics in bladder cancer tissues and paracancerous tissues.Cell invasion and migration experiments showed that MAFG-AS1,ASMTL-AS 1,LINC02321 and LINC00922 can significantly affect the invasion and migration of bladder cancer cells.Conclusion:Our study showed that SMALLL signature can be used as an important indicator for the prognosis of patients with bladder cancer.These lncRNAs are related to extracellular matrix network,and have great differences in immune cell infiltration and molecular subtypes among different risk groups,which are promising to become biological targets for precise treatment of bladder cancer patients.