| Objective:Triple negative breast cancer(TNBC)is the most complex molecular subtype in breast cancer.It does not express any target gene in the other three types of breast cancer molecular typing,so it has attracted much attention.TNBC accounts for 10-20% of breast cancer,most of them are young women,with high metastasis rate,high histological grade and easy recurrence.The disease mechanism of TNBC is still unclear,and clinical diagnosis and treatment are difficult,The purpose of this project is to:1.Through bioinformatics method,the differentially expressed genes of TNBC,normal breast tissue and non TNBC tumor tissue were screened,the biological functions of TNBC were analyzed,the enriched cell components and the main signal pathways were located,the genes related to TNBC prognosis were predicted,and the expression ofsignificant differentially expressed genes in breast cancer tissues and cell lines were analyzed,so as to determine The core genes that may affect the occurrence and development of TNBC.2.By observing the expression of key genes Nek2 and ?-catenin in TNBC tumor tissues and cell lines,and analyzing the relationship between them and the clinical characteristics of TNBC patients,thereby digging new theoretical basis for TNBC clinical diagnosis and providing reliable biomarkers Thing.Methods:1.Analysis of genes related to the development of TNBC by bioinformatics(1)Screen differentially expressed genes in TNBC and non-TNBC tumor tissues and normal breast tissues(1)Select the two gene chips GSE38959(TNBC vs normal breast)and GSE27447(TNBC vs non-TNBC)from the Gene Expression Omnibus(GEO)database,use the GEO2 R online analysis tool to analyze the differentially expressed genes(DEG)in GSE38959 and GSE27447,and then P value <0.05,| log2(fold-change)| ?2 is used as the cutoff value to select meaningful DEG.(2)The meaningful DEGs in GSE38959 and GSE27447 are intersected through Venny2.1 online software,and a common DEG is selected for further analysis.(2)Analysis of biological functions of differentially expressed genes in GSE38959 and GSE27447(1)All the meaningful DEGs and common DEGs were tested in FunRich3.1.3software for their molecular functions,biological processes involved,enriched cellular components and involved signaling pathways.(3)Screening of TNBC core genesIn the Kaplan-Meier plotter,the common DEG associated with the prognosis ofTNBC patients was screened and used as the core gene of TNBC for further analysis.(4)Bioinformatics analysis of Nek2 expression in tumor tissues and cell lines(1)Use the ARCHS4 database to analyze the expression of Nek2 in different cell lines.(2)Analyze the expression of Nek2 in different tumor tissues using GEPIA database.2.The clinical significance of the selected core genes Nek2 B and ?-Catenin and their expression in TNBC tissues and cell lines(1)TNBC cell lineCulture human TNBC cell lines Hs578 T,BT20,MDA-MB-231 and MDA-MB-468(purchased from Chinese Academy of Medical Sciences)(2)Detect the expression of Nek2 B and ?-catenin in TNBC cell lineWestern-blot and qRT-PCR were used to detect the expression of Nek2 B and?-catenin in TNBC cell line.(3)Expression of Nek2 B and ?-catenin in TNBC tumor and its clinicopathological relationshipThe tissue samples of 80 TNBC patients in the pathology department of the second hospital of Shanxi Medical University from 2007 to 2012 and the corresponding clinical pathological data were collected,and the tissue chips were made.The expression of Nek2 B and ?-Catenin protein and the relationship between clinical pathological characteristics were detected by immunohistochemistry.Results:1.Bioinformatics analysis of DEG in TNBC(1)Screening and function of DEGWe obtained 998 eligible DEG,including 563 down-regulated genes and 423up-regulated genes;423 up-regulated DEG mainly enriched cell components in nucleosome,molecular function in DNA binding,biological process in chromosome separation,and 563 down-regulated DEG mainly enriched extracellular matrix;998 DEG mainly participated in cell cycle / mitosis,DNA replication and M-M / G1 stage of mitosis And M period.(2)Determination and function of core genesAfter taking the intersection of the two data sets,a total of 13 common differentially expressed genes were obtained for Nek2,THSD4,ESR1,TFF1,AGR3,RNF128,FOXA1,WIF1,PRR15,AZGP1,PMCHL1,LAMP3,and ANKRD30A;13 DEGs with common expression differences Mainly involved in Wnt / ?-catenin signaling pathway;survival analysis of 13 common differentially expressed genes showed that only Nek2 and TNBC patients were significantly related to disease-free survival and relapse-free survival(P <0.05),and the differential expression was the largest,so Nek2 As a core gene for further analysis.(3)Bioinformatics analysis of Nek2 expression in TNBCIn the ARCHS4 database,the expression of Nek2 in triple-negative breast cancer cells is higher than the average level compared to cell lines in other parts;in the GEPIA database,Nek2 is expressed in breast cancer tissues compared with tumor tissues in other parts Has the highest expression.2.Expression of Nek2 B and ?-catenin in TNBC tumor tissues and cell lines and their relationship with clinical characteristics(1)Expression of Nek2 B and ?-catenin in TNBC cell line and tumor tissueThe protein expression of Nek2 B and ?-catenin in TNBC cells was analyzed by Western blot.The results showed that both were highly expressed in Hs578 t and BT20 cell lines,while both were low expressed in MDA-MB-231 and MDA-MB-468 cell lines.Next,the same result was verified by qRT-PCR.(2)Relationship between Nek2 B and clinicopathological characteristicsIn TNBC patient specimens,53 patients(66.3%)expressed Nek2 B.The high expression of Nek2 B is associated with the high histological staging(G3)group(P<0.001),the lymph node metastasis group(P = 0.022)and the high Ki-67 positive index group(P = 0.014).Higher expression rate than its negative cases(50/69,72.5%,P =0.018).Conclusion:1?Through biological information analysis,the occurrence and development of TNBC may be related to mitosis and the Wnt/?-catenin signaling pathway.Nek2 as a core gene is highly expressed in breast cancer tumor tissues and cell lines,and is significantly related to the poor prognosis of TNBC2?The high expression of Nek2 B is related to the poor prognosis of TNBC patients.In TNBC tumor tissues and cells,the expression of Nek2 B is positively correlated with the expression of ?-catenin,suggesting that Nek2 B may affect the occurrence and development of TNBC by regulating Wnt / ?-catenin signaling pathway. |
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