{'fraction_non_alphanumeric': 0.056843115743280305, 'fraction_numerical': 0.03695831047723533, 'mean_word_length': 5.016914191419142, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Circular RNA ABCB10 promotes cell proliferation and invasion, but inhibits apoptosis via regulating the microRNA-1271-mediated Capn4/Wnt/β-catenin signaling pathway in epithelial ovarian cancer

Circular RNA ABCB10 (circ-ABCB10) modulates cellular functions and microRNA (miR)-1271 in epithelial ovarian cancer (EOC). The present study aimed to investigate the interaction between circ-ABCB10 and miR-1271 in regulating EOC cellular function and the calpain small subunit 1 (Capn4)/Wnt/β-catenin signaling pathway. circ-ABCB10 and miR-1271 expression levels were detected in EOC cells (OVCAR3, UWB1.289, SKOV3 and CAOV3) and normal ovarian epithelial cells (IOSE80) via reverse-transcription quantitative PCR. SKOV3 cells were transfected with control short hairpin (sh)RNA plasmids, control inhibitor, circ-ABCB10 shRNA plasmids and miR-1271 inhibitor. UWB1.289 cells were transfected with control overexpression plasmids, control mimic, circ-ABCB10 overexpression plasmids and miR-1271 mimic. Subsequently, cell proliferation, apoptosis, invasion and the Capn4/Wnt/β-catenin signaling pathway were assessed. In addition, a luciferase activity assay was performed. circ-ABCB10 expression was significantly increased in OVCAR3, SKOV3 and CAOV3 cells compared with IOSE80 cells, but was not significantly altered in UWB1.289 cells. miR-1271 expression was significantly decreased in OVCAR3, UWB1.289, SKOV3 and CAOV3 cells compared with IOSE80 cells. In both SKOV3 and UWB1.289 cells, circ-ABCB10 negatively regulated miR-1271, whereas miR-1271 did not affect circ-ABCB10. Furthermore, circ-ABCB10 enhanced cell proliferation, invasion and the Capn4/Wnt/β-catenin signaling pathway, but inhibited cell apoptosis, whereas miR-1271 suppressed cell proliferation, invasion and the Capn4/Wnt/β-catenin signaling pathway, but facilitated cell apoptosis. Moreover, miR-1271 attenuated the proproliferative, proinvasive and antiapoptotic effects of circ-ABCB10, and reversed the positive regulation of circ-ABCB10 on the Capn4/Wnt/β-catenin signaling pathway. Besides, the luciferase activity assay indicated that circ-ABCB10 directly bound to miR-1271. In conclusion, the present study indicated that circ-ABCB10 promoted cell proliferation and invasion, and suppressed apoptosis by regulating the miR-1271-mediated Capn4/Wnt/β-catenin signaling pathway in EOC.


Introduction
Epithelial ovarian cancer (EOC) accounts for ~90% of ovarian cancer cases, which is the most common cause of gynaecological cancer-related death, causing >150,000 deaths annually worldwide (1,2). Due to the asymptomatic nature of EOC at early stages and the lack of an effective screening program for assessing disease risk, patients are generally diagnosed at an advanced stage (International Federation of Gynecology and Obstetrics stage III and IV), which is associated with the dissemination of cancer cells from the ovary to the peritoneal cavity (1,3,4). Surgical resection and platinum-based chemotherapy are the standard treatment strategies for patients with EOC (4). However, the majority of patients develop chemoresistance and recurrence, which results in a poor 5-year relative survival rate (5). To improve the prognosis in patients with EOC, identifying the molecular pathogenies underlying EOC is important for the development of novel and effective drug targets.
Circular RNA (circ/circRNA)-ABCB10, a novel circRNA, exerts oncological effect in several types of cancer, such as breast and non-small-cell lung cancer (6)(7)(8). As for EOC, our previous study revealed that circ-ABCB10 correlates with deteriorated clinical features and poor prognosis in patients with EOC. Moreover, the previous study indicated that circ-ABCB10 facilitates cell proliferation, inhibits cell apoptosis and negatively regulates microRNA (miR)-1271, miR-1252 and miR-203 in EOC cells (9). Meanwhile, miR-1271 suppresses cell proliferation and invasion by downregulating calpain small subunit 1 (Capn4), and Capn4 enhances EOC cell proliferation and migration by targeting the Wnt/β-catenin signaling pathway (10,11). Based on the aforementioned studies, it was hypothesized that circ-ABCB10 might competitively interact with miR-1271 to promote EOC progression by activating Capn4 and its downstream Wnt/β-catenin signaling pathway. Therefore, the present study aimed to investigate the interaction between circ-ABCB10 and miR-1271 in regulating cell proliferation, apoptosis, invasion and the Capn4/Wnt/β-catenin signaling pathway in EOC.
Cell proliferation, apoptosis and invasion. Cell proliferation was assessed at 0, 24, 48 and 72 h post-transfection using a Cell Counting Kit-8 assay (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. At 48 h post-transfection, cell apoptosis was assessed using an Annexin V-FITC Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. Apoptotic cells (early and late apoptosis) were detected via flow cytometry using CytoFLEX system (Beckman Coulter, Inc.), and FlowJo software 7.6 (FlowJo LLC) was used for data analysis. In addition, at 48 h post-transfection, cell invasion was evaluated by performing a Transwell assay. Each Transwell insert (size, 8 µm; Corning, Inc.) was pre-coated with Matrigel (at 37˚C for 1 h), then, SKOV3 (3x10 4 cells) and UWB1.289 cells (3x10 4 cells) suspended in serum-free culture medium (Gibco; Thermo Fisher Scientific, Inc.) were added into the upper chamber (Corning). Culture medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added into the lower chamber. After 24 h of incubation, cells that invaded the Matrigel coated filters were fixed with methanol (Sigma-Aldrich; Merck KGaA), stained with crystal violet (at 37˚C for 15 min; Sigma-Aldrich; Merck KGaA) and counted with a BX 41 inverted microscope under x200 magnification (Olympus Corporation).
Statistical analysis. Data are presented as the mean ± standard deviation of three experimental repeats. Comparisons between two groups were analyzed using the unpaired Student's t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Dunnett's or Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed by SPSS 22.0 Software (IBM Corp.), and graphs were plotted by GraphPad Prism 7.02 Software (GraphPad Software, Inc.).
circ-ABCB10 and miR-1271 expression levels following transfection. In SKOV3 cells, circ-ABCB10 expression levels were significantly decreased in the circ (-) group compared with the NC (-) group, but were similar between the miR (-) group and the NC (-) group, as well as between the circ (-) + miR (-) and circ (-) groups ( Fig. 2A). miR-1271 expression levels were significantly decreased in the miR (-) group compared with the NC (-) group, and in the circ (-) + miR (-) group compared with the circ (-) group. By contrast, miR-1271 expression levels were significantly increased in the circ (-) group compared with the NC (-) group (Fig. 2B). In UWB1.289 cells, circ-ABCB10 expression levels were significantly increased in the circ (+) group compared with NC (+) group, but similar between the miR (+) group and the NC (+) group, as well as between the circ (+) + miR (+) group and the circ (+) group (Fig. 2C). Furthermore, miR-1271 relative expression levels were significantly elevated in the miR (+) group compared with the NC (+) group, and in the circ (+) + miR (+) group compared with the circ (+) group. By contrast, miR-1271 expression was significantly decreased in the circ (+) group compared with the NC (+) group (Fig. 2D). Collectively, the results indicated that circ-ABCB10 negatively regulated miR-1271, whereas miR-1271 did not regulate circ-ABCB10 in EOC.
circ-ABCB10 promotes cell proliferation by targeting miR-1271. In SKOV3 cells, cell proliferation was significantly increased in the miR (-) group compared with NC (-) group at 48 and 72 h. Similarly, cell proliferation was significantly increased in the circ (-) + miR (-) group compared with circ (-) group at 48 and 72 h, but significantly decreased in the circ (-) group compared with the NC (-) group at 48 and 72 h (Fig. 3A). In UWB1.289 cells, cell proliferation was significantly reduced in the miR (+) group compared with the NC (+) group at 72 h, and in the circ (+) + miR (+) group compared with the circ (+) group at 48 and 72 h. By contrast, cell proliferation was significantly enhanced in the circ (+) group compared with the NC (+) group at 48 and 72 h (Fig. 3B). Collectively, the results   indicated that miR-1271 reversed the proproliferative effect of circ-ABCB10 in EOC.

circ-ABCB10 inhibits cell apoptosis by targeting miR-1271.
In SKOV3 cells, cell apoptosis was significantly decreased in the miR (-) group compared with the NC (-) group, and in the circ (-) + miR (-) group compared with the circ (-) group. By contrast, cell apoptosis was significantly increased in the circ (-) group compared with the NC (-) group (Fig. 4A and B). In UWB1.289 cells, cell apoptosis was significantly increased in the miR (+) group compared with the NC (+) group, and in the circ (+) + miR (+) group compared with the circ (+) group. Moreover, cell apoptosis was significantly reduced in the circ (+) group compared with the NC (+) group (Fig. 4C and D). Collectively, the results suggested that miR-1271 attenuated the antiapoptotic effect of circ-ABCB10 in EOC.

circ-ABCB10 promotes cell invasion by targeting miR-1271.
In SKOV3 cells, cell invasion was significantly increased in the miR (-) group compared with the NC (-) group, and in the circ (-) + miR (-) group compared with the circ (-) group. By contrast, cell invasion was significantly decreased in the circ (-) group compared with the NC (-) group (Fig. 5A and B). In UWB1.289 cells, cell invasion was significantly decreased in the miR (+) group compared with the NC (+) group, and in the circ (+) + miR (+) group compared with the circ (+) group, but significantly increased in the circ (+) group compared with the NC (+) group (Fig. 5C and D). Collectively, the results indicated that miR-1271 reversed the proinvasive effect of circ-ABCB10 in EOC.

Discussion
EOC is a highly heterogeneous malignancy that is characterized by different precursor lesions, tissues of origin, molecular properties and clinical outcomes (2,4). Despite advances in molecular predictors (for example, cancer antigen 125 and human epididymal protein 4), surgical debulking and chemotherapy for EOC in the past three decades, the clinical outcomes remain undesirable with increased chemoresistance and recurrence in patients with EOC (2,13). To develop a novel and effective treatment strategy, the molecular pathogenesis underlying EOC requires further investigation.
With the application of high-throughput technologies, the underlying mechanisms and applications of circRNAs as therapeutic targets are widely explored in various diseases (14). circRNA, a covalently closed non-coding RNA, is generated by non-random back-splice events, is resistant to RNA degradation and has limited protein coding ability (15). circRNAs either function as oncogenic stimuli or tumor suppressors in cancer development and progression by sponging miRNAs to regulate the activities of downstream target genes or protein expression (6,15). circ-ABCB10 is involved in the tumorigenesis of various types of cancer (6)(7)(8).
In breast cancer, circ-ABCB10 knockdown inhibits breast cancer cell proliferation but facilitates apoptosis by interacting with miR-1271 (7). In non-small cell lung cancer, circ-ABCB10 expression is upregulated in NSCLC cell lines, which enhances cell proliferation and migration via sponging miR-1252 to upregulate forkhead box 2 (FOXR2) synthase expression (8). As for EOC, our previous study uncovered that circ-ABCB10 expression is elevated in EOC cell lines compared with normal ovarian epithelial cells, and it increases cell proliferation, but decreases apoptosis and the expression of miR-1271, miR-1252 and miR-203 (9). miR-1271 serves as a tumor suppressive miRNA, which inhibits cell proliferation and invasion via modulating downstream target genes in various types of cancer (10,16,17). In EOC, miR-1271 inhibits cell viability, invasion and epithelial mesenchymal transition by directly binding to the 3'-untranslated region of zinc finger E-box binding homeobox 1 mRNA in EOC (17). In colorectal cancer, miR-1271 promotes cell proliferation and invasion by suppressing Capn4 (10). Capn4 facilitates cell proliferation and migration in EOC by activating the Wnt/β-catenin signaling pathway (11). Therefore, it was hypothesized that circ-ABCB10 might promote the progression of EOC via targeting miR-1271, Capn4 and the Wnt/β-catenin signaling pathway.
In the present study, the results indicated that circ-ABCB10 expression was upregulated, whereas miR-1271 expression was downregulated in EOC cells compared with normal ovarian cells. To investigate the role of the interaction between circ-ABCB10 and miR-1271 in cell proliferation, invasion and apoptosis, rescue experiments were performed. circ-ABCB10 negatively regulated miR-1271, whereas miR-1271 did not regulate circ-ABCB10 in EOC. Furthermore, circ-ABCB10 enhanced cell proliferation and invasion, but inhibited cell apoptosis in EOC. By contrast, miR-1271 suppressed cell proliferation and invasion, and facilitated cell apoptosis in EOC. miR-1271 reversed the proproliferative, proinvasive and antiapoptotic effects of circ-ABCB10 in EOC. Collectively, the results suggested that circ-ABCB10 facilitated cell proliferation and invasion, but suppressed cell apoptosis by negatively regulating miR-1271 in EOC. A few potential explanations for the results of the present study include: i) circ-ABCB10 might affect the function of its host gene ABCB10, which facilitates EOC tumor progression; and ii) circ-ABCB10 inhibited miR-1271 expression, which downregulated its downstream proteins (e.g. Capn4, FOXR2, regulator of G-protein signaling 17 and spindlin 1), resulting in enhanced tumor growth, metastasis and invasion of EOC (8,10,18,19). The results also indicated that circ-ABCB10 increased Capn4 expression by repressing miR-1271.
In conclusion, this study indicated that circ-ABCB10 promoted cell proliferation and invasion, but suppressed apoptosis by regulating the miR-1271-mediated Capn4/Wnt/β-catenin signaling pathway in EOC, highlighting the involvement of the circ-ABCB10/miR-1271/Capn4/Wnt/β-catenin signaling pathway in the progression of EOC.