Abstract
BackgroundIncreasing studies have shown that circRNA is closely related to the carcinogenesis and development of many cancers. However, biological functions and the underlying molecular mechanism of circRNAs in triple-negative breast cancer (TNBC) remain largely unclear so far.MethodsHere, we investigated the expression pattern of circRNAs in four pairs of TNBC tissues and paracancerous normal tissues using RNA-sequencing. The expression and prognostic significance of circSEPT9 were evaluated with qRT-PCR and in situ hybridization in two TNBC cohorts. The survival curves were drawn by the Kaplan-Meier method, and statistical significance was estimated with the log-rank test. A series of in vitro and in vivo functional experiments were executed to investigate the role of circSEPT9 in the carcinogenesis and development of TNBC. Mechanistically, we explored the potential regulatory effects of E2F1 and EIF4A3 on biogenesis of circSEPT9 with chromatin immunoprecipitation (ChIP), luciferase reporter and RNA immunoprecipitation (RIP) assays. Furthermore, fluorescent in situ hybridization (FISH), luciferase reporter and biotin-coupled RNA pull-down assays were implemented to verify the relationship between the circSEPT9 and miR-637 in TNBC.ResultsIncreased expression of circSEPT9 was found in TNBC tissues, which was positively correlated with advanced clinical stage and poor prognosis. Knockdown of circSEPT9 significantly suppressed the proliferation, migration and invasion of TNBC cells, induced apoptosis and autophagy in TNBC cells as well as inhibited tumor growth and metastasis in vivo. Whereas up-regulation of circSEPT9 exerted opposite effects. Further mechanism research demonstrated that circSEPT9 could regulate the expression of Leukemia Inhibitory Factor (LIF) via sponging miR-637 and activate LIF/Stat3 signaling pathway involved in progression of TNBC. More importantly, we discovered that E2F1 and EIF4A3 might promote the biogenesis of circSEPT9.ConclusionsOur data reveal that the circSEPT9 mediated by E2F1 and EIF4A3 facilitates the carcinogenesis and development of triple-negative breast cancer through circSEPT9/miR-637/LIF axis. Therefore, circSEPT9 could be used as a potential prognostic marker and therapeutical target for TNBC.
Highlights
Increasing studies have shown that circRNA is closely related to the carcinogenesis and development of many cancers
In the top 15 upregulated circRNAs, we focused on a novel circRNA, circSEPT9, which was generated from exon2 of SEPT9 gene by back-splicing (645 bp) on the basis of the annotation of circBase, its back splicing junction was validated by Sanger sequencing and the presence of circSEPT9 was proved by RT-PCR (Fig. 1b)
The results revealed that ectopic expression of miR-637 significantly attenuated the proliferation, migration and invasion-promoting effects induced by up-regulation of circSEPT9 in triplenegative breast cancer (TNBC) cells, while miR-637 inhibitors could counteract the inhibitory roles of circSEPT9 knockdown in proliferation, migration and invasion of TNBC cells by EdU, colony formation, wound healing and transwell assays (Fig. 7a-e)
Summary
Increasing studies have shown that circRNA is closely related to the carcinogenesis and development of many cancers. Biological functions and the underlying molecular mechanism of circRNAs in triplenegative breast cancer (TNBC) remain largely unclear so far. Breast cancer is the most common cause of cancer-related mortality in women around the world. Triple-negative breast cancer (TNBC) is a subgroup of breast cancer, which is defined as lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This subgroup comprises around 15~20% of all breast cancers [1]. It is urgent to find feasible molecular target for TNBC
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