Abstract
BackgroundRecent studies have revealed that circular RNAs (circRNAs) play significant roles in the occurrence and development of many kinds of cancers including breast cancer (BC). However, the potential functions of most circRNAs and the molecular mechanisms underlying progression of BC remain elusive.MethodHere, Circular RNA microarray was executed in 4 pairs of breast cancer tissues and para-cancer tissues. The expression and prognostic significance of circACTN4 in BC cells and tissues were determined by qRT-PCR and in situ hybridization. Gain-and loss-of-function experiments were implemented to observe the impacts of circACTN4 on the growth, invasion, and metastasis of BC cells in vitro and in vivo. Mechanistically, chromatin immunoprecipitation, luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation, fluorescence in situ hybridization and co-immunoprecipitation assays were executed.ResultsCircACTN4 was significantly upregulated in breast cancer tissues and cells, its expression was correlated with clinical stage and poor prognosis of patients with BC. Ectopic expression of circACTN4 strikingly facilitated the growth, invasion, and metastasis of breast cancer cells in vitro and in vivo. Whereas knockdown of circACTN4 revealed opposite roles. CircACTN4 was mainly distributed in the nucleus. Further mechanistic research proved that circACTN4 could competitively bind to far upstream element binding protein 1 (FUBP1) to prevent the combination between FUBP1 and FIR, thereby activating MYC transcription and facilitating tumor progression of breast cancer. Furthermore, we found that upstream transcription factor 2 (USF2) might promote the biogenesis of circACTN4.ConclusionOur findings uncover a pivotal mechanism that circACTN4 mediated by USF2 might interact with FUBP1 to promote the occurrence and development of breast cancer via enhancing the expression of MYC. CircACTN4 could be a novel potential target for diagnosis and treatment of breast cancer.
Highlights
Recent studies have revealed that circular RNAs play significant roles in the occurrence and development of many kinds of cancers including breast cancer (BC)
CircACTN4 could only be amplified by divergent primers from cDNA but not gDNA. d The localization of circACTN4 was investigated in BC cells and tissues with Fluorescence in Situ Hybridization (FISH). e Nuclearcytoplasmic fractionation assay displayed that circACTN4 was mostly distributed in the nucleus of BC cells
U6 was considered as a nuclear control and GAPDH was used as a cytoplasmic protein control. f and g The expressions of circACTN4 and ACTN4 of BC cells were analyzed by qRT-PCR and RT-PCR after treatment with actinomycin D. h The levels of circACTN4 and ACTN4 were detected after RNase R digestion at different time points by qRT-PCR. i RT-PCR was used to detect the expression of circACTN4 and ACTN4 after with RNase R treatment. j Schematic model of wild type (WT) and mutant (Mut) sequences of two putative binding sites of upstream transcription factor 2 (USF2) on ACTN4 promoter
Summary
Recent studies have revealed that circular RNAs (circRNAs) play significant roles in the occurrence and development of many kinds of cancers including breast cancer (BC). Breast cancer is the malignant tumor with the highest incidence among women in the world. The early detection and effective systematic treatment of breast cancer have improved, it remains the leading cause of cancer death in women all over the world [1]. Circular RNAs (circRNAs) are a new class of RNAs that have been widely found in various species through high-throughput sequencing in recent years. Recent researches have demonstrated that circRNAs have important potential roles in regulating gene expression. CircRNAs exert their functions, including as miRNA sponges, RNA-binding protein scaffolds, intermediates in RNA alternative splicing (AS), protein translation templates and transcription regulators [4]. The interaction of circRNAs and proteins were less studied in human cancers
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