Study On Differentially Expressed Genes In Breast Cancer Based On Microarray, RNA-Sequencing Data And CMAP Database

Objective:(1)Based on microarray and RNA-sequencing data,breast cancer gene expression profile datasets were analyzed to obtain differentially expressed genes to investigate the possible molecular mechanisms involved in breast cancer and provide clues for finding breast cancer-related biomarkers.(2)To study the expression of Collagen Type VI Alpha 6 Chain(COL6A6),Thrombospondin 2(THBS2)and Collagen Type V Alpha 1 Chain(COL5A1)in breast cancer tissues and adjacent tissues,and to explore the relationship between their expression and the clinicopathological features and prognosis of breast cancer.(3)By comparing breast cancer differential genes with Connectivity Map(CMAP)databases,the small molecular compounds that are negatively related with opposite breast cancer gene expression were screened in order to find potential drugs that could be used to interfere with the development and treatment of breast cancer.Materials and Methods:(1)The gene expression profiles of breast cancer were collected and calculated from the Gene Expression Omnibus(GEO)database and The Cancer Genome Atlas(TCGA)database to obtain differentially expressed genes(DEGs)in breast cancer.Protein-protein interaction(PPI)networks and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed to explore the molecular mechanisms of breast cancer.(2)The RNA expression levels of the target genes COL6A6,THBS2,and COL5A1 in the GEO database were validated to analyze their potential diagnostic value.The protein expression of COL6A6,THBS2 and COL5A1 in 136 breast cancer tissues and 55 paracancerous tissues was further examined by immunohistochemical SupervisionTM two-step method.The relationship between their expression and clinicopathological features of breast cancer patients and their prognostic significance was explored.(3)By comparing the breast cancer differential genes with the CMAP database website,10 negatively related small molecule compounds with the greatest correlation with breast cancer were retrieved,combined with literature searches to analyze the potential and mechanism of breast cancer treatment.Result:(1)The DEGs of breast cancer were obtained from the GEO database and the TCGA database.A total of 1,525 differentially expressed genes were obtained after the crossover,of which 965 genes were down-regulated and 560 genes were up-regulated.The GO analysis showed that these DEGs were mainly enriched in biological processes such as extracellular matrix organization,negative regulation of angiogenesis,cell adhesion,cell division.KEGG analysis showed that these DEGs are mainly located in 26 pathways such as ECM-receptor interaction,Cell cycle,Focal adhesion.The ECM-receptor interaction pathway was the most significant,and there were 27 DEGs in this pathway.We selected COL6A6,THBS2 and COL5A1 as the target gene for our study.(2)The meta-analysis of the expression levels of COL6A6,THBS2,and COL5A1 in breast cancer in the GEO database showed that COL6A6 is low in breast cancer [mean standard deviation(SMD)=-2.09,95% confidence interval(CI):-2.33 ~-1.86,p=0.073];THBS2 is highly expressed in breast cancer(SMD=0.71,95%CI: 0.37~1.06,P=0.000);COL5A1 is highly expressed in breast cancer(SMD=0.71,95%CI: 0.51 ~ 0.92,P=0.255).The area under the SROC curve(AUC)for COL6A6,THBS2 and COL5A1 was 0.95(95% CI: 0.93 ~ 0.97),0.85(95% CI: 0.82 ~ 0.88),0.84(95% CI: 0.80 ~ 0.87),respectively.The expression of COL6A6,THBS2 and COL5A1 in 136 cases of breast cancer tissues and 55 cases of adjacent normal tissues were detected by immunohistochemistry.Low expression rate of COL6A6 was 81.6% in 136 breast cancer tissues.The high expression and low expression of COL6A6 protein in cancer and adjacent tissues were statistically significant(P=0.002).There was a significant difference in the expression and distant metastasis of COL6A6 protein between breast cancer tissues(P=0.000).There was no significant difference in the expression of COL6A6 protein in breast cancer tissues with age,histological grade,tumor size,lymph node metastasis,clinical stage and molecular typing,and the expression of ER,PR,HER-2,P53 and Ki-67.(all P>0.05).Kaplan-Meier survival analysis showed that there was a significant difference between the low expression,high expression and survival rate of COL6A6 protein(P=0.047).The high expression rates of THBS2 and COL5A1 in breast cancer were 91.9% and 90.4%,respectively.There was no statistically significant correlation between the expression of THBS2 and COL5A1 and the relationship between their expression and clinical pathological features and prognosis.(3)The top ten small molecule compounds that most negatively related with breast cancer DEGs were screened through the CMAP database.They were 15-delta prostaglandin J2,fulvestrant,medrysone,phenoxybenzamine,resveratrol,pyrvinium,hesperetin,sirolimus,Prestwick-559,and chlorpromazine,respectively.They are small-molecule compounds that have a reverse effect on differential gene expression in breast cancer.Some of these drugs are already classic drugs for the treatment of breast cancer.Some small-molecule compounds have not yet been applied in the treatment of breast cancer and may become new potential therapeutic drugs for breast cancer patients.Conclusion:(1)The use of computational biology methods can effectively screen and analyze the gene expression profiles of breast cancer in public databases to obtain DEGs,providing a theoretical basis for studying the pathogenesis of breast cancer at the molecular level.(2)Low expression of COL6A6 gene in breast cancer may promote tumor progression and distant metastasis,and may have a certain prognostic value.The level of THBS2 expression at the RNA level also has a certain prognostic value.Although the two genes of THBS2 and COL5A1 are highly expressed in breast cancer tissues,there is no obvious relationship between their expression and clinical pathological features.The potential value of THBS2 and COL5A1 remains to be further explored and verified by large-scale clinical trials.(3)Based on the differential genes of breast cancer,the CMAP database can be used to screen potential small molecule compounds for breast cancer treatment.