circGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a

He et al. Journal of Experimental & Clinical Cancer Research circGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a Rongfang He 0 2 3 Peng Liu 1 Xiaoming Xie 1 Yujuan Zhou 4 Qianjin Liao 4 Wei Xiong 0 3 Xiaoling Li 0 3 Guiyuan Li 0 3 Zhaoyang Zeng 0 3 Hailin Tang 1 0 The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, Xiangya Hospital, Central South University , Changsha, Hunan , China 1 Department of Breast Oncology, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center of Cancer Medicine , Guangzhou , China 2 Department of Pathology, The First Affiliated Hospital of University of South China , Hengyang, Hunan Province , China 3 The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University , Changsha, Hunan , China 4 Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University , Changsha, Hunan , China Backgroud: Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in triple negative breast cancer (TNBC) is largely unknown. Methods: We performed circRNA microarrays to identify circRNAs that are aberrantly expressed in TNBC cell lines. Expression levels of a significantly upregulated circRNA, circGFRA1, was detected by quantitative real-time PCR (qRTPCR) in TNBC cell lines and tissues. Kaplan-Meier survival analysis was used to explore the significance of circGFRA1 in clinical prognosis. Then, we examined the functions of circGFRA1 in TNBC by cell proliferation, apoptosis and mouse xenograft assay. In addition, luciferase assay was used to explore the miRNA sponge function of circGFRA1 in TNBC. Results: Microarray analysis and qRT-PCR verified a circRNA termed circGFRA1 that was upregulated in TNBC. KaplanMeier survival analysis showed that upregulated circGFRA1 was correlated with poorer survival. Knockdown of circGFRA1 inhibited proliferation and promoted apoptosis in TNBC. Via luciferase reporter assays, circGFRA1 and GFRA1 was observed to directly bind to miR-34a. Subsequent experiments showed that circGFRA1 and GFRA1 regulated the expression of each other by sponging miR-34a. Conclusions: Taken together, we conclude that circGFRA1 may function as a competing endogenous RNA (ceRNA) to regulate GFRA1 expression through sponging miR-34a to exert regulatory functions in TNBC. circGFRA1 may be a diagnostic biomarker and potential target for TNBC therapy. Circular RNAs; miR-34a; GFRA1; Competitive endogenous RNAs; Triple negative breast cancer Background Breast cancer is the most commonly diagnosed cancer in women worldwide. It is estimated that there will be 255,180 new cases and 41,070 deaths of breast cancer in the United States in 2017 [ 1 ]. The last few decades have witnessed outstanding advances in breast cancer treatment. However, the prognosis for triple negative breast cancer (TNBC) remains poor. Therefore, it is significant to develop more effective therapeutic strategies to treat breast cancer, especially TNBC. Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely expressed in mammals [ 2 ]. A plenty of circRNAs have been identified, but their potential functions are poorly understood. There are currently few reports describing the role of circRNAs in breast cancer. Liang G et al. reported that circDENND4C is a HIF1αassociated circRNA promoting the proliferation of breast cancer under hypoxia [ 3 ]. Lu L et al. provided a profile of circRNAs in breast cancer and adjacent normal-appearing tissues [ 4 ]. However, the function of circRNAs in TNBC progression is unclear. Revealing the role of circRNAs will be critical for understanding TNBC pathogenesis and offering a novel insight into identificating new biomarkers or therapeutic targets of TNBC. microRNAs (miRNAs) are endogenous, non-proteincoding, single-stranded 19- to 25-nucleotide RNAs that play a vital role in the process of cancer [ 5 ]. miR-34a has been reported to act as a tumor suppressor to regulate tumor progression and is always down-regulated in cancers [ 6 ], including prostate cancer [ 7 ], glioblastoma [ 8 ], colon cancer [ 9 ] and breast cancer [ 10–12 ]. Due to the significant role that miR-34a plays in cancer, development of miR-34a-based gene therapy is encouraged for multiple types of cancers. It is reported that RNAs can act as competitive endogenous RNAs (ceRNAs) to co-regulate each other by competing for shared microRNAs [ 13, 14 ]. Studies by several groups have illustrated that mRNAs, pseudogenes, long noncoding RNAs (lncRNAs) and circRNAs may all serve as ceRNAs [15]. circRNAs in mammals have also been shown to function as miRNA sponges or ceRNAs. Memczak (...truncated)