Screening Of Differential Expressed Genes In Breast Cancer And Research Of Its Function And Mechanism

OBJECTIVE: To explore the molecular mechanisms of development in the breast cancer,this study used microarray data from the GEO database to screen differential expressed genes between breast cancer tissues and normal breast tissues,providing a theoretical basis for early diagnosis and clinical treatment of breast cancer.Methods: Three groups of breast cancer gene expression profile microarray data were downloaded from the public gene database GEO database.The differential expressed genes between breast cancer tissue and normal breast tissue were screened by R software.The differential expression was multiplied by more than 1 time and P value was < 0.05.GO enrichment analysis,KEGG pathway analysis and STRING protein interaction analysis were performed by the differential expressed genes.Online analysis on Kaplan-Meier Plotter(http://kmplot.com/analysis/)to select genes which have a significant effect on breast cancer survival and prognosis and Online analysis the expression of the selected genes in TCGA breast and normal tissues through UALCAN(http://ualcan.path.uab.edu/).The expression of tissues,the functional experiments that proliferation,apoptosis,invasion,migration,and the related mechanisms of the selected genes in breast cancer cell lines were performed,and animal models were established.RESULTS: In the first part,according to the three sets of GEO expression profiles of breast cancer,the differential expressed genes between breast cancer tissues and normal tissues were analyzed,and there were 1334 differential genes with overlapping genes,of which 358 were up-regulated and 976 genes were down-regulated expression.54 terms were obtained by enrichment analysis of the differential expressed genes.The first 10 terms were GO:1901681 sulfur compound binding,GO:0048037 cofactor binding,GO:0016614oxidoreductase activity,acting on CH-OH group of donors,GO:0050662 coenzyme binding,GO:0016616 oxidoreductase activity,acting on the CH-OH group of donors,NAD or NADP as acceptor,GO:0003779 actin binding,GO:0005539 glycosaminoglycan binding,GO:0033218 amide binding,GO:0008201 heparin binding,GO:0051287 NAD binding.KEGG pathway analysis differential expressed genes are mainly enriched in 7 signaling pathways which is glycolysis/gluconeogenesis,citric acid cycle(TCA cycle),ascorbic acid and metabolism,fructose and mannose metabolism,pentose phosphate pathway,galactose metabolism,pent Mutual conversion of sugar and glucuronic acid.All the differential genes were input into the STRING database,and the protein interaction analysis of all genes was carried out.The top 10 genes were FN1,IL6,MYC,CDH1,CDK1,JUN,CCNB1,FGF2,CCNA2,and EZH2.The Pubmed and K-M survival analysis was used to screening down-regulated genes affecting survival and prognosis of breast cancer,and gene OGN(Osteoglycin)and ITM2A(Integral membrane protein 2A)were found.Furthermore,it was further verified by TCGA database that the expression levels of OGN and ITM2 A in Luminal,Her-2 positive and triple negative breast cancer were lower than those in normal tissues.In the second part,OGN and ITM2 A were found significantly under-expressed in breast cancer tissues.The breast cancer cell lines were transfected with plasmids over-expressing OGN and ITM2 A respectively.After transfection of OE-OGN with MCF-7and MDA-MB-231 cells,the clonal formation of the cells was inhibited,and the ability of invasion and migration of MDA-MB-231 cells was weakened.The mechanism was detected by western blot.Epithelial-like E-cadherin expression of Epithelial-mesenchymal transition(EMT)associated proteins were elevated,and mesenchymal proteins Vimentin and N-cadherin were lost.The most important transcription factors of EMT,Snail1 and ZEB1 were lost.Loss of p-Akt and loss of p-m TOR after overexpression of OGN could been found.After remediation experiments with 740Y-P which was the PI3K/Akt pathway activator,p-Akt,p-m TOR,N-cadherin,and Snail1 were up-regulated.The plasmids of OE-ITM2 A were transfected into MCF-7 and MDA-MB-231 cells.The proliferation of CCK8 and colony formation were inhibited,the apoptosis was increased,and the ability of invasion and migration of MDA-MB-231 cells was weakened.The intrinsic apoptosis pathway-related proteins were detected by western blot.The apoptosis proteins BCL-2 and MCL-1 which inhibited cell apoptosis were down-regulated,and the intrinsic apoptosis proteins Cytochrome C,Caspase 3,Cleaved caspase 3,and PARP1 were up-regulated.In the third part,it was confirmed by the establishment of a nude mouse animal model that the tumor-forming magnetic resonance imaging of the breast cancer cell line MCF-7was significantly reduced in nude mice by transfecting the plasmid expressing OGN or transfecting the plasmid expressing ITM2 A.The tumor tissues of nude mice were taken out,embedded in paraffin,and histochemical staining of tumor tissues showed that the expression levels of OGN and ITM2 A were higher in the subcutaneous tumor tissues of nude mice up-expressed OGN and ITM2 A than in the control group.Conclusion: The expression levels of OGN and ITM2 A were under-expressed in breast cancer tissues.The gene OGN could inhibits EMT of breast cancer through the PI3K/Akt/m-TOR pathway,thereby inhibiting invasion and migration of breast cancer cells in vitro and tumor formation in vivo.The gene ITM2 A promotes the intrinsic apoptosis of breast cancer cells and inhibits the proliferation of breast cancer cells in vitro and tumor formation in vivo.