Effect Of Transcription Factor E2F5 On Triple Negative Breast Cancer And Its Related Mechanism

Background and PurposeBreast cancer is one of the most common malignancies in women worldwide.In the four molecular subtypes of breast cancer,triple negative breast cancer(TNBC)is considered to be the most severe subtype with the worst prognosis.As a series of transcription factor,E2F family play key roles in multiple biological processes,and many cell cycle related proteins are regulated by E2F.It was reported that the aberrant expression of E2F was strongly associated with tumorigenesis and progression.The present study evaluated the expression level of E2F family,and picked out E2F5 as the potential bio-target of breast cancer.The aim was to reveal the bio-function of E2F5,and to explore its possible regulatory mechanism through a series of assays in vitro and in vivo.Materials and MethodsReal time PCR and IHC was employed to detect the expression level of E2F family in 10 breast cancer and adjacent normal tissues,and validated by public data from TCGA and GEO database.Chi-square test was used to analysis the correlation between E2F5 expression and clinicopathological features of breast cancer.Moreover,Kaplan-Miere analysis and Cox regression were employed to reveal the correlation between E2F5 expression and breast cancer prognosis.Subsequently,two E2F5overexpressed cell lines,MDA-MB-231 and MDA-MB-468 were established by lentivirus stable transfection,and two E2F5 knock-down cell lines,SUM159 and BT549 were established by si RNA transient transfection.A series of in vitro and in vivo assays were performed to investigate the effect of E2F5 expression on cell proliferation,migration,invasion,and tumor stem cell properties.RT-PCR was used to estimate the expression level of genes associated with drug resistance and epithelial-mesenchymal transition(EMT)of TNBC in E2F5 knocked down cell lines.Finally,in E2F5 knock-down cell lines,ZEB2 was knocked down by si RNA transfection,and the tumor stem cell properties was evaluated to understand the association between ZEB2 down regulation and E2F5.ResultsAccording to the result of q RT-PCR in breast cancer and normal samples,The expression of E2F family was up-regulated in cancerous tissues,and the analysis of TCGA and GEO public data showed similar results.E2F5 was picked out as potential target gene of breast cancer,and IHC showed that E2F5 protein expression was higher in cancerous tissues rather than adjacent normal tissues.The analysis on the correlation between E2F5 and disease features showed that,patients with high E2F5expression attended to have later clinical stage and more lymph node metastasis,and a larger proportion of triple negative breast cancer.Kaplan-Miere and Cox regression analysis showed that E2F5 high expression was strongly associated with poor prognosis in breast cancer,especially in TNBC.CCK-8,colony formation and tumorigenesis assays indicated that E2F5 improved the proliferation of TNBC cell lines.Transwell and sphere formation assays showed that E2F5 over-expression was associated with stronger invasion ability and stem cell properties.Subsequently,IC₅₀of TNBC cell lines was tested by CCK-8 assay,and it was observed that E2F5knock-down was associated with stronger drug sensitivity.RT-PCR showed that E2F5induced EMT in TNBC cell lines.ZEB2 was the potential down-stream target gene of E2F5.ZEB2 knock-down could antagonize the improvement of stem cell properties caused by E2F5.ConclusionE2F5 was up-regulated in cancerous tissue,and was associated with tumorigenesis,progression and metastasis of breast cancer.E2F5 could improve the tumor stem cell properties of TNBC cell lines,and could enhance the resistance of antineoplastic drugs.ZEB2 was the potential down-stream target gene of E2F5.E2F5could up-regulate the expression of ZEB2,and finally cause the effect mentioned above.