Function And Molecular Mechanism Of CircSET9 Mediated By E2F1 And EIF4A3 In The Carcinogenesis And Development Of Triple-negative Breast Cancer

Objective More and more studies have shown that circRNA are closely related to the occurrence and development of many cancers.The purpose of this study was to investigate the expression,biological function and molecular mechanism of circSEPT9 in human triple-negative breast cancer(TNBC),and to analyze regulation mechanism of its expression,so as to provide scientific basis for its clinical diagnosis and treatment of a new molecular target in TNBC.Methods RNA sequencing(RNA-seq)was performed to obtain the differentially expressed circRNA expression profiles by analyzing 4 pairs of TNBC and para-cancer normal tissues.CircSEPT9 was identified by Sanger sequencing,PCR amplification,RNase R enzyme digestion and actinomycin D treatment.The expression of circSEPT9 in TNBC patients was detected by in situ hybridization and quantitative real-time PCR(qRT-PCR),and the relationship between the expression of circSEPT9 and clinicopathological characteristics was analyzed,and the prognosis was further evaluated.A series of in vivo and in vitro functional experiments were conducted to investigate the role of circSEPT9 in the carcinogenesis and development of TNBC.In terms of mechanism,chromatin immunoprecipitation(ChIP),luciferase reporter gene and RNA immunoprecipitation(RIP)were executed to analyze the potential regulatory effects of E2F1 and EIF4A3 on the biogenesis of circSEPT9.In addition,bioinformatics analysis,luciferase reporter,biotin-coupled RNA pull-down,fluorescence in situ hybridization(FISH),western blot and immunohistochemistry(IHC)were implemented to investigate whether circSEPT9 could regulate tumor-related signaling pathways bybinding to miR-637.Results The expression of circSEPT9 in TNBC tissues and cells was significantly higher than that in para-carcinoma tissues and normal breast epithelial cells,and its expression level was positively correlated with clinical advanced stage and poor prognosis.Functional experiments demonstrated that down-regulation of circSEPT9 could inhibit the growth and metastasis of TNBC cells in vivo and in vitro,and induce TNBC cell apoptosis and autophagy.However,the up-regulation of circSEPT9 exerted opposite effects.Further experimental studies showed that circSEPT9 could regulate the expression of Leukemia inhibitory Factor(LIF)by targeting miR-637,and further activated the LIF/Stat3 signaling pathway.In addition,we found that E2F1 and EIF4A3 might promote the biogenesis of circSEPT9.Conclusion CircRNA circSEPT9,which is mediated by E2F1 and EIF4A3,can bind to miR-637 to promote the expression of LIF and activate the LIF/Stat3 signaling pathway,thus promoting the occurrence and development of triple-negative breast cancer.Therefore,circSEPT9 can be used as a potential biomarker and therapeutic target for TNBC.