Abstract
BackgroundRecent studies indicate that circular RNA (circRNA) plays a pivotal role in cancer progression. Here, we sought to investigate its role in breast cancer.MethodsCircANKS1B (a circRNA originated from exons 5 to 8 of the ANKS1B gene, hsa_circ_0007294) was identified by RNA-sequencing and validated by qRT-PCR and Sanger sequencing. Clinical breast cancer samples were used to evaluate the expression of circANKS1B and its associations with clinicopathological features and prognosis. Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to support clinical findings and elucidate the function and underlying mechanisms of circANKS1B in breast cancer.ResultsCircANKS1B was significantly up-regulated in triple-negative breast cancer (TNBC) compared with non-TNBC tissues and cell lines. Increased circANKS1B expression was closely associated with lymph node metastasis and advanced clinical stage and served as an independent risk factor for overall survival of breast cancer patients. Functional studies revealed that circANKS1B promoted breast cancer invasion and metastasis both in vitro and in vivo by inducing epithelial-to-mesenchymal transition (EMT), while had no effect on breast cancer growth. Mechanistically, circANKS1B abundantly sponged miR-148a-3p and miR-152-3p to increase the expression of transcription factor USF1, which could transcriptionally up-regulate TGF-β1 expression, resulting in activating TGF-β1/Smad signaling to promote EMT. Moreover, we found that circANKS1B biogenesis in breast cancer was promoted by splicing factor ESRP1, whose expression was also regulated by USF1.ConclusionsOur data uncover an essential role of the novel circular RNA circANKS1B in the metastasis of breast cancer, which demonstrate that therapeutic targeting of circANKS1B may better prevent breast cancer metastasis.
Highlights
Recent studies indicate that circular RNA plays a pivotal role in cancer progression
We found that compared with si-NC, ESRP1 knockdown dramatically decreased circANKS1B production in breast cancer cells transfected with wild-type or individually mutation circANKS1B minigenes, whereas had little or no effect on circANKS1B production in cells transfected with a/b or c/d mutation minigenes (Fig. 6d), suggesting that these four motifs on flanking introns are necessary for ESRP1-mediated circANKS1B biogenesis
The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes cell invasion and metastasis via inducing TGF-β1-mediated epithelial-tomesenchymal transition (EMT) in breast cancer we found that the increased migratory and invasive capabilities of MCF-7 cells caused by circANKS1B overexpression could be extensively retarded by ESRP1 or USF1 knockdown or miR-148a/152-3p overexpression, as well as by treatment with LY2109761 (TGF-β receptor type I/II inhibitor), as demonstrated by wound healing, transwell migration and invasion assays (Fig. 7a-b)
Summary
Recent studies indicate that circular RNA (circRNA) plays a pivotal role in cancer progression. Accumulating evidence shows that epithelial-tomesenchymal transition (EMT) is the pivotal step for breast cancer cells to metastasis, whereby epithelial cells gradually lose polarity and adhesion capacity but gain mesenchymal traits with down-regulation of the epithelial biomarker. It is widely accepted that TGF-β signaling is a primary EMT inducer by activation of Smad complexes that translocate into the nucleus to regulate gene expression [7], which is critical for breast cancer progression and heterogeneity [8]. Growing studies showed that circRNAs were involved in various cancer biological processes, including EMT and metastasis, via interaction with miRNA [17, 18]. Despite advancements in the study of circRNAs, the potential correlation between circRNAs and breast cancer metastasis is still unclear and remains to be further investigated
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