Mechanism Of E2F1 Promotes The Malignant Progression In Triple Negative Breast Cancer

[Background]Breast cancer(BC)is the tumor with the highest morbidity and mortality rate among women in the world.Triple negative breast cancer(TNBC)is characterized by complex disease progression,high degree of malignancy,rapid metastasis and poor prognosis,which has become a major obstacle to clinical treatment.Therefore,to clarify the pathogenesis of TNBC and to search for early specific markers,it has become an urgent need to conquer TNBC.Abnormal expression of E2F1 can be involved in a variety of tumor malignant phenotypes.Previous studies have shown that E2F1 interacts with microRNAs(miRNAs)to promote tumor progression.E2F1-regulated miR-17-5p is involved in several signaling pathways that regulate tumor epithelial mesenchymal transition,impair tumor cell autophagy,and induce relapse.In addition,F Box and Leucine-rich Repeat Protein 5(FBXL5),a target gene downstream of miR-17-5p,is closely related to iron metabolism and is involved in the malignant phenotype and progression of a variety of tumors.Although recent studies have shown that miRNA-mediated FBXL5 is associated with tumorigenesis and development,the mechanism of miR-175p/FBXL5 regulation by E2F1 in TNBC and its effects have not been reported.[Purpose]The aim of this study was to investigate the role and mechanism of E2F1 in TNBC progression and to provide a new laboratory evidence for prognosis adjudgement of TNBC patients.[Methods]1.The expression level of E2F1 in BC was analyzed by TCGA database.Normal mammary epithelial cell lines(MCF-10A)and BC cell lines(MDA-MB-231,BT549,T47D,MCF-7)were cultured,and the expression level of E2F1 in BC cell lines was measured by real-time quantitative polymerase chain reaction(qRT-PCR)technology.2.The TNBC cell lines were infected with knockdown/overexpression E2F1 lentivirus,and the cell lines with stable low expression/overexpression of E2F1 were screened by puromycin.The infection efficiency was detected by qRT-PCR and Western blot(WB).The miR-17-5p mimic and miR-17-5p mimic NC were transfected with TNBC cell lines using liposomes,and the expression of miR-17-5p in the cells was detected by qRT-PCR.3.The proliferation ability of TNBC cells was detected by CCK-8 assay,clone formation assay and EdU assay.The migration ability of TNBC cells was detected by scratch assay.The invasion ability of TNBC cells was detected by Transwell assay.Cell cycle was analyzed by flow cytometry.4.MiRDB,ONCOMIR,TARGRTSCAN,TCGA online website were used to predict the downstream target genes of miR-17-5p,qRT-PCR and WB were used to detect the expression of miR-17-5p downstream target genes in TNBC cells.The direct relationship between miR-17-5p and downstream target genes was verified by dual luciferase reporter assay;The expression level of the target gene FBXL5 was detected by WB.[Results]1.The results by TCGA database analysis and qRT-PCR showed that E2F1 was significantly highly in BC cells and in TNBC cells(MDA-MB-231 and BT549)compared to normal epithelial cells of the breast,which were significantly different from other BC cells(p<0.001).2.In vitro functional assays showed that the proliferation,migration and invasion of tumor cells were inhibited after knockdown of E2F1,and cell G1/S phase transformation was reduced,while the opposite was true for overexpression of E2F1.3.The expression level of miR-17-5p decreased after knockdown of E2F1;Dual luciferase reporter gene assay showed that miR-17-5p directly bound to the 3’UTR region of E2F1 and inhibits the expression of E2F1.E2F1 interacts with miR-17-5p to regulate and promoted the malignant progression of TNBC.4.Online database as well as dual luciferase reporter gene assay confirmed that FBXL5 was a downstream target gene of miR-17-5p,and TCGA correlation analysis revealed that the expression level of FBXL5 was significantly negatively correlated with miR17-5p(r=-0.5651,p<0.001).[Conclusions]E2F1 and miR-17-5p form a negative feedback regulatory loop in TNBC cells,and miR-17-5p directly targets and regulates FBXL5,which plays an important role in promoting malignant progression of TNBC.