Key Words Gas5 • MiR-23a • ATG3 • CeRNA • Breast cancer • Autophagy Abstract Background/Aims: An increasing body of evidence shows that long noncoding RNAs (lncRNAs) are involved in many different cancers. In this study, we aimed to investigate the competing endogenous RNA (ceRNA)-dependent mechanism by which the lncRNA GAS5 contributes to the development of breast cancer. Methods: A total of 68 breast cancer patients were enrolled, and breast cancer and adjacent normal tissues were collected.

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... he human breast cancer cell lines MDA-MB-231, MDA-MB-453, BT549, SK-BR-3 and MCF-7 and human breast cell line MCF10A were utilized in this study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were performed to detect expression of relative factors. RNA immunoprecipitation (RIP) was used to evaluate the relationship between GAS5 and miR-23a, and a dual luciferase reporter gene assay was employed to assess the relationship between ATG3 and miR-23a. A subcutaneous xenograft nude mouse model was generated to examine the role of GAS5 and its regulatory pathway in autophagy. Results: GAS5 levels were frequently decreased in breast cancer tissues and cell lines, and its relatively low expression was closely related to a larger tumour size, advanced tumour-node-metastasis (TNM) stage and estrogen receptor-negative (ER-) breast cancer tissues. More importantly, we found that GAS5 promoted autophagy, with enhanced autophagosome formation after GAS5 overexpression. GAS5 was found to act as a microRNA sponge in a pathway that included miR-23a and its target gene ATG3. The GAS5-miR-23a-ATG3 axis significantly regulated autophagy in vivo and in vitro. Conclusions: In summary, we report that the GAS5-miR-23a-ATG3 axis can be regarded as a key regulator of autophagy pathways in breast cancer; it may constitute a promising biomarker and therapeutic target in the future. Introduction Breast cancer is the most commonly diagnosed type of malignancy and the second leading cause of cancer-related death among women [1, 2] . Breast cancer usually manifests as a lump in the breast with common symptoms that include pain/soreness in the breast region, skin puckering or dimpling, a rash around the breast area, discharge from the nipple, and a change in breast shape [3] . Breast cancer is devastating disease in both developed and developing countries, is the most prevalent cancer in Chinese women, and accounts for approximately 12.2% of new cancer cases and 9.6% of deaths in worldwide [4] . The development of breast cancer is associated with many factors, including age, overweight status, smoking, drinking, reproduction, physical inactivity, family history of breast disease, recent use of oral contraceptives, and radiation exposure [5, 6] . Neoadjuvant and systemic chemotherapy are widely applied for treatment of breast cancer, whereas successful molecular-targeted therapies of any type are not yet unavailable [7, 8] . Therefore, there is an urgent need to find new therapeutic targets to improve the survival rate of patients with breast cancer. Long non-coding RNAs (lncRNAs), a class of RNAs over 200 nucleotides in length, have been found to play key roles in the important secondary structures and the specific binding that participate in multiple gene regulatory networks [9, 10] , and several studies report that many types of lncRNAs have crucial functions in tumourigenesis, including breast cancer [11] [12] [13] . Recently, a new regulatory circuit was discovered that involves RNA competition crosstalk for shared microRNAs (miRNAs), whereby competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules to their targets and thus impose an additional level of post-transcriptional regulation [14] . Previous studies have also revealed the interactive relationship between lncRNAs and miRNAs in many diseases and cancers, including glioma and prostate cancer [15] [16] [17] . Located at 1q25, growth arrest-specific transcript 5 (GAS5), which was originally isolated from mouse NIH 3T3 cells, is a noncoding, small nucleolar RNA host gene [18, 19] . As a stress-inducible gene, the tumour-suppressive role of GAS5 has been reported [20] , and decreased expression of GAS5 was found in breast cancer in association with a poor prognosis [21] . GAS5 directly interacts with miR-23a to negatively regulate its expression [22] . In a previous study, we also found that lncRNA-GAS5 can act as a miR-23a sponge. It has been reported that miR-23a expression is significantly higher in breast cancer patients with lymph node metastasis compared with those without lymph node metastasis [23] . Furthermore, ATG3, an E2-like enzyme that catalyses ATG8 phosphatidylethanolamine conjugation, which is essential for autophagy, is the target of miR-23a; indeed, ATG genes conserved from yeast to humans are required for autophagy [24, 25] . In this study, we aimed to determine the role of the lncRNA GAS5-miR-23a-ATG3 axis in the regulation of autophagy in patients with breast cancer. Materials and Methods Ethics statement All experiments in our study conformed to clinical research guidelines, were approved by the Ethics Committee of Nanjing Medical University and were conducted in strict accordance with animal experiment rules according to the Declaration of Helsinki. All study participants provided written informed consent. Sample collection A total of 68 eligible patients who were histologically diagnosed with breast cancer and underwent surgical resection at Wuxi Clinical Hospital of Nanjing Medical University between 2014 and 2016 were enrolled in our study. None of the patients received chemotherapy or radiotherapy. Breast cancer tissue and adjacent normal tissue samples were obtained, and pathological stages were evaluated by an experienced pathologist who had no authorship of this study. The staging of patients was assessed according to the International Union Against Cancer's (UIAC's) tumour-node-metastasis (TNM) system. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C for further use.