Circular RNA circABCC4 as the ceRNA of miR‐1182 facilitates prostate cancer progression by promoting FOXP4 expression

Abstract In recent years, circular RNAs (circRNAs) have been identified to be essential regulators of various human cancers. However, knowledge of the functions of circRNAs in prostate cancer remains very limited. The correlation between circABCC4 and human cancer is largely unknown. This study aims to investigate the biological functions of circABCC4 in prostate cancer progression and illustrate the underlying mechanism. We found that circABCC4 was remarkably up‐regulated in prostate cancer tissues and cell lines and promoted FOXP4 expression by sponging miR‐1182 in prostate cancer cells. CircABCC4 knockdown markedly suppressed prostate cancer cell proliferation, cell‐cycle progression, migration and invasion in vitro. Furthermore, silencing of the circRNA also delayed tumor growth in vivo. Taken together, our findings indicated that circABCC4 facilitates the malignant behaviour of prostate cancer by promoting FOXP4 expression through sponging of miR‐1182. The circABCC4–miR‐1182‐FOXP4 regulatory loop may be a promising therapeutic target for prostate cancer intervention.

This study investigates the biological function of circABCC4 in promoting prostate cancer progression. CircABCC4 (circBase ID: hsa_circ_0030586; chr13:95813442-95840796) was derived from the mRNA back-splicing of ABCC4, which is located in chromosome 13q32.1. CircRNA array analysis showed that a novel circRNA named circABCC4 is highly up-regulated in prostate cancer tissues. Moreover, high circABCC4 expression is associated with a poor prognosis in prostate cancer patients. Loss-or gain-of-function analyses indicated that circABCC4 promotes prostate cancer progression in vitro and in vivo.
Collectively, our findings reveal that circABCC4 plays a pivotal role in prostate cancer progression.

| Patient specimens
A total of 47 cases of prostate cancer tumor tissues and paired adjacent normal samples was collected from the Second Xiangya Hospital, Central South University. The procedure of the present study was approved by the Institutional Review Board of the Second Xiangya Hospital, Central South University. Written informed consent was obtained from each participant prior to their participation in this research.

| Cell viability assay
A total of 4 × 10 3 cells were seeded into 96-well plates and then incubated for 24 hours. The cells were then transfected with the indicated RNA duplexes, treated with 10 μL of cell counting solution (WST-8, Dojindo Laboratories, Tokyo, Japan) at different time points, and incubated for another 2 hours. The absorbance of the wells was measured spectrophotometrically at 450 nm.

| Colony formation assay
A total of 500 transfected cells were seeded into 6-well plates and cultured for 14 days. The cells were subsequently fixed with methanol and stained with 0.1% crystal violet. Then, the colony number was counted.

| Flow cytometry analysis
A total of 1 × 10 6 cells were harvested and fixed with ice-cold 75% ethanol overnight. The cells were then incubated with 50 μg/ mL propidium iodide (Invitrogen, CA, USA) and 50 μg/mL RNaseA (Sigma, St. Louis, MO, USA) for 45 minutes at room temperature.
Thereafter, the samples were analysed using an Arial III FACS system (BD Biosciences, USA).

| Luciferase assays
After plating on a 24-well plate for 24 hours, PC3 and DU145 cells were transfected with the indicated transcripts using Lipofectamine 3000 (Invitrogen) and cultured for another 24 hours. Relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA).

| qRT-PCR
Total RNA was extracted using RNAiso Plus (TaKaRa, Japan), and cDNA was synthesized using the PrimeScript RT Reagent Kit

| Western blot analysis
Cells were lysed with lysis buffer (20 mM KCl, 150 mM NaCl, 1% NP-40, 1% Triton X-100, 50 mM NaF, 50 mM Tris, 1 mM DTT, 1 mM EGTA, 1 × protease inhibitor and 10% glycerol) for 1 hour and centrifuged for 30 minutes at 4°C. Equal amounts of protein were separated on SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated first with primary antibodies and then with secondary antibodies. Signals were detected using an ECL kit (Bio-Rad).

| Statistical analyses
All statistical data are expressed as the mean ± SD of at least three independent experiments. Two-tailed Student's t test and one-way ANOVA were used to compare two and multiple groups, respectively. The Kaplan-Meier method was used to draw survival curves and the log-rank test was used to determine statistical significance.
Pearson's correlation analysis was used to determine the correlations. A P value of <0.05 was considered to indicate statistical significance.

| Relative expression of circABCC4 in prostate cancer tissues
Although circRNAs play indispensable roles in the development of cancers, 15 their functions in prostate cancer have not been fully investigated. Thus, circRNA expression was analysed from a public prostate cancer database (GSE77661) to reveal the possible role of circRNAs in prostate cancer carcinogenesis. Many circRNAs were differentially expressed in prostate cancer, among which circABCC4 was remarkably overexpressed ( Figure 1A). CircABCC4 was also overexpressed in both prostate cancer tumors and cell lines compared with the control ( Figure 1B, 1), thereby confirming that cir-cABCC4 is overexpressed in prostate cancer. The expression of circABCC4 is correlated with prostate cancer carcinogenesis because higher circABCC4 expression indicates shorter 5-year survival rates among patients ( Figure 1D). Moreover, we found that the expression of circABCC4 was positively correlated with advanced stage and metastasis (Table 1). These findings collectively indicate that circABCC4 may be involved in prostate cancer.

| CircABCC4 deficiency inhibits prostate cancer cell proliferation, migration and invasion
Functional assays were carried out to investigate the function of circABCC4 in prostate cancer progression and carcinogenesis.
Because circABCC4 levels were the highest in PC3 and DU145 cell lines ( Figure 1C), we chose them for further investigation.

| CircABCC4 promotes prostate cancer propagation in vivo
The aforementioned experiments show that circABCC4 promotes prostate cancer progression in vitro. Nude mice were injected with circABCC4-deficient and control cells, and tumor growth was

| CircABCC4 is a sponge of miR-1182
CircRNAs can function via miRNA binding. 16 The possible binding partners of circABCC4 were predicted to explore the mechanism of circABCC4 in prostate cancer. An AGACCCUC motif in circABCC4 was found to match that of miR-1182 ( Figure 4A), thereby indicating the possibility that circABCC4 binds to miR-1182. Luciferase assays showed that circABCC4 does bind to miR-1182 ( Figure 4B), and mutation of the AGACCCUC motif blocks the circABCC4-miR-1182 interaction ( Figure 4A, 4). Knockdown results showed that circABCC4 deficiency up-regulates miR-1182 expression ( Figure 4C), which means the circABCC4-miR-1182 interaction could inhibit miR-1182 expression. Indeed, the expressions of circABCC4 and miR-1182 were negatively correlated ( Figure 4D), and miR-1182 was downregulated in prostate cancer tissues ( Figure 4E). These results are in line with the observed circABCC4 overexpression in prostate cancer and reveal that circABCC4 is a sponge of miR-1182.

| FOXP4 is a target of miR-1182
MiRNAs can bind to and inhibit mRNA expression. 17 To further understand the mechanism of miR-1182 in prostate cancer progression, the binding partner of miR-1182 was predicted, and results showed that miR-1182 may bind to FOXP4 mRNA ( Figure 5A). Luciferase experiments confirmed the binding of miR-1182 to FOXP4 mRNA ( Figure 5B). Furthermore, miR-1182 overexpression in PC3 and DU145 cells remarkably inhibited FOXP4 expression ( Figure 5C, 5D), which is in line with the negative expression correlation of miR-1182 and FOXP4 ( Figure 5E). As expected, FOXP4 expression was elevated in prostate cancer tissues ( Figure 5F). Thus, miR-1182 binds to FOXP4 and inhibits its expression, which means FOXP4 is a target of miR-1182.

| CircABCC4 regulates prostate cancer progression by modulating miR-1182/ FOXP4 signalling
From the results above, we can confirm that circABCC4 and FOXP4 are overexpressed whereas miR-1182 is down-regulated in prostate cancer. CircABCC4 also sponges miR-1182 expression, whereas miR-1182 negatively regulates FOXP4 expression. Studies have shown a circRNA-miRNA-mRNA regulation axis in cancer progression. 18 Thus, we sought to determine whether circABCC4 promotes prostate cancer progression via miR-1182/FOXP4 signalling. First, we analysed whether circABCC4 regulates FOXP4 expression and found that circABCC4 deficiency in PC3 and DU145 cells could markedly inhibit the expression of FOXP4 ( Figure 6A, 6B, 6D). In addition, overexpression of circABCC4 in miR-1182-overexpressing PC3 and DU145 cells could rescue the inhibitory effect of miR-1182 on FOXP4 expression ( Figure 6A, 6), which means circABCC4 promotes FOXP4 expression through miR-1182. MiR-1182 deficiency in cir-cABCC4-deficient PC3 and DU145 cells remarkably rescued FOXP4 expression ( Figure 6D). These results demonstrate that circABCC4 regulates FOXP4 expression through miR-1182. CircABCC4 expression appears to be positively correlated with FOXP4 expression in prostate cancer tissues ( Figure 6C), further indicating that cir-cABCC4 regulates FOXP4 expression.
Next, we sought to discover whether circABCC4 promotes prostate cancer progression through miR-1182/FOXP4 signalling.
We inhibited miR-1182 expression and restored FOXP4 expression

| D ISCUSS I ON
CircRNAs are known to be involved in the progression of many cancers. 14,15,19 Previous studies have shown that circRNAs are dysregulated in many cancers. 20

| CON CLUS ION
Taking the results together, we showed that circABCC4 expression is elevated in prostate cancer and revealed that the circRNA functions as an oncogene. CircABCC4 mechanically sponges miR-1182 expression, resulting in the up-regulation of FOXP4 and prostate cancer progression. We further discovered a critical cir-cABCC4-miR-1182-FOXP4 axis in prostate cancer progression and demonstrated the important role of circRNAs in the progression of this disease. However, our study also has a limitation. Because we performed bioinformatics analysis to screen out circABCC4 only using a pair of tumor tissues and normal control, other important circRNAs may be ignored.

ACK N OWLED G EM ENTS
None.

CO N FLI C T S O F I NTE R E S T
All authors declare that they have no conflicts of interest.

AUTH O R S CO NTR I B UTI O N
CH and XZ initiated this study and designed the experiments. CH performed experiments and analysed the data. HD, YW, HJ, RX, XZ F I G U R E 6 CircABCC4 regulates prostate cancer progression by modulating miR-1182/FOXP4 signalling. A, B, Overexpression of circABCC4 abrogated the effect of the miR-1182 mimic on FOXP4 expression in PC3 and DU145 cells, whereas mutation of the binding site in circABCC4 abolished this trend. C, Correlation between circABCC4 and FOXP4 expression in prostate cancer tissues. D, Protein levels of FOXP4 at the indicated cell lines. E, Cell proliferation was measured by CCK8 assay. F, G, Cell migration and invasion was evaluated by transwell assay of the indicated cell lines. *P < 0.05 and ZH analysed the data. XZ wrote the manuscript. All authors read the manuscript and approved it.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.