Downregulation of circ-TRPS1 suppressed prostatic cancer prognoses by regulating miR-124-3p/EZH2 axis-mediated stemness

Background: Abnormal circular RNA (circRNA) expression correlates with human traits such as many kinds of cancers. Though circRNAs have links to cancer, they have less characterization in metastatic castration-resistant prostate cancer (PCa), which is main reason for PCa mortality. Methods: Therefore, high-throughput sequencing was used for selected circRNA proles. We performed RT-qPCR to determine circ-TRPS1 expression regarding PCa tissues. We used uorescence in situ hybridization (FISH) to detect circ-TRPS1 expression and circ-TRPS1 subcellular localization in PCa tissues. circ-TRPS1 expression in PCa cells was selectively regulated. We employed CCK8, transwell assays to monitor the cell proliferation and invasion, respectively. We employed dual-luciferase reporter and RNA pulldown assays to verify the relationship among circ-TRPS1, miR-124-3p, and EZH2. We examined the circ-TRPS1 effects on PCa cell metastasis and proliferation in vivo through a subcutaneous xenograft model as well as an intravenous tail injection model of nude mice. Results: The result showed that circ-TRPS1 was upregulated signicantly in high-grade PCa tissues or cell lines. High circ-TRPS1 expression correlated to aggressive PCa phenotypes. Knockdown of circ-TRPS1 suppressed PCa proliferation and metastasis through targeting miR-124-3p/EZH2 axis-mediated stemness in PCa, which was validated by luciferase reporter assays. EZH2 overexpression or miR-124-3p inhibition reversed the inhibition of circ-TRPS1 silencing in PCa cell migration and proliferation by recovering stemness. Conclusion: In summary, data demonstrated that circ-TRPS1 suppressed PCa progression through functioning similar to a miR-124-3p stem-like cell


Background
Prostate cancer (PCa) is amongst the most popular carcinomas in male. Organ-con ned PCa is e ciently treated through radical prostatectomy [1]. For advanced PCa, androgen deprivation therapy is regarded as rst-line treatment [2]. If hormone resistance occurs, advanced PCa is usually severe because of PCa metastasis [3]. Therefore, studying molecular mechanisms and characterizing new targets for the development of therapeutics for patients with PCa is indispensable.
Although some circRNA involvement is reported in PCa, investigations into biological mechanism and regulatory functions regarding circRNAs in PCa remain largely unclear. In this manner, the regulatory mechanisms regarding circRNAs in cancer need to be elucidated. This investigation studied circRNA expression pro les in different grades of PCa tissues. We identi ed that circ-TRPS1 was increased signi cantly in high grade-PCa tissues and was related closely to PCa prognosis. We also discovered that circ-TRPS1 might act as an antioncogene sponge of miR-124-3p to enhance EZH2 expression and promote cancer stem-like cell-mediated PCa progression. As a result, circ-TRPS1 increases could function as biomarkers for prognosis and prediction, and a candidate PCa therapeutic target.

Cell culture and transfection
We cultured PC3, LnCaP, and DU145 cells. We maintained them in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with penicillin of 100 units/ml and streptomycin of 100 µg/ml. We maintained RWPE-1 cells in K-SMF medium (Gibco, Grand Island, NY, USA) supplemented with epidermal growth factor of 5 ng/mL and bovine pituitary extract of 50 µg/mL. We incubated cultures in humidi ed environment with CO 2 of 5% and 37 °C.
Tumor sphere formation assays We harvested PC3 and LnCaP cells, and resuspended them as single cells in serum-free medium. After cell counting, we added 200 cells/well in 200 µL serum-free medium to 96-well plates, 10 wells per group, changing medium every 2 days. We obtained images of ve randomly selected regions for each group of wells with a camera-equipped microplate reader (Leica, Wetzlar, Germany) and calculated sphere percentage as number of spheres/200.

Cell proliferation assays
We employed Cell Counting Kit-8 assays (CCK-8; Gibco, Logan, Utah, USA) to quantify cellular proliferation. We seeded transfected cells into plates with 96 wells with 5,000 cells/well in triplicate, and determined cell viability with a CCK-8 system at 0, 24, 48 and 72 h after seeding, following given procedures.
For colony formation assays, we seeded cells that transfected into plates with 6 wells at 2,000 cells/well and maintained them in 1640 containing FBS of 10% for ten days. We imaged and counted colonies after xing and staining.

Transwell assays
For invasion assays, we placed Transwell assay inserts (Millipore, Billerica, MA, USA) into plates with 24 wells, coating membranes in upper chambers with Matrigel (BD Biosciences). We placed 500 µL 1640 containing 10% FBS into bottom chambers and 10,000 cells in 200 µL serum-free 1640 in upper chambers. After 1 ~ 2 days, we utilized methanol to x cells on membranes and stained them with crystal violet before observing them via microscope (Leica).

Western blots
We extracted protein from tissues or cells with RIPA lysis buffer and performed western blots as previously described [15]. Primary antibodies were against OCT4, Nanog and GAPDH (Cell Signaling Technology, Beverly, MA, USA) and blots were stained following standard procedures. We visualized immunoreactivity with chemiluminescence detection kits (Western Blotting Substrate, Donghuan Biotech, China).
For metastasis analysis, we transfected 2 × 10 5 NC or circ-TRPS1-silenced PC3 cells with luciferase expression vectors, and injected cells intravenously into mouse tails. After 30 d, PC3 cell metastases were analyzed by bioluminescence imaging following an intravenous injection of luciferin (150 mg luciferin/kg body weight) into tails.

Immunohistochemistry
We xed tumor tissue samples in formalin 10% and embedded them in para n. We stained sections (5µm) with Ki67 to explore proliferation. We examined sections with an Axiophot light microscope and imaged them via digital camera.

Statistics analyses
We assessed differences among groups via paired/unpaired two-tailed t-tests using Pearson's correlation tests to obtain associations among groups. We denoted data by mean ± SEM. P-values < 0.05 indicated signi cant. We performed all statistical analysis with GraphPad Prism (GraphPad Inc., San Diego, CA, USA).

Results
High Circ-TRPS1 expression predicted unfavorable prognoses for PCa To determine correlations between circRNA expression and PCa progression, three PCa samples from H-PCa and three from L-PCa were used in circRNA high-throughput sequencing. The results showed a series differentially expressed circRNA in PCa tissues comparing (Fig. 1A). RT-qPCR illustrated the six upregulated circRNA expressions in H-PCa and L-PCa tissues. The results showed that hsa_circ_0006950 (circ-TRPS1) had the highest expression in H-PCa tissues comparing with L-PCa tissues (Fig. 1B). RT-qPCR also showed that circ-TRPS1 expression increased in H-PCa tissues compared to L-PCa patients (Fig. C). FISH assays demonstrated that circ-TRPS1 expression increased in H-PCa tissues compared with L-PCa tissues. Circ-TRPS1 was localized predominantly to the cytoplasm (Fig. 1D). To further understand the prognostic value of circ-TRPS1 expression, we analyzed correlations with patient characteristics. High circ-TRPS1 expression correlated with higher pathological T stage, lymph node metastasis, and distant metastasis ( Table 1). Circ-TRPS1 is derived from circularizing exons from gene TRPS1, which are at chr8:116631358-116635985. TRPS1 is 4627 bp and its spliced mature circRNA is 1087 bp (Fig. 1E). The ndings indicated that circ-TRPS1 functioned in PCa progression. Downregulation of circ-TRPS1 suppressed PCa proliferationin vivo and in vitro To identify circ-TRPS1 functions in PCa proliferation, we employed RT-qPCR to detect circ-TRPS1 expression. Circ-TRPS1 expression increased in PCa cell lines PC3, LnCaP, and DU145 compared with RWPE-1 ( Fig. 2A). PC3 and LnCaP cells had the highest circ-TRPS1 levels, so they were selected for further experiments. Circ-TRPS1 expression decreased signi cantly in both PC3 and LnCaP cells after transfection with an siRNA against circ-TRPS1 (Fig. 2B). CCK8 (Fig. 2C and 2D) and colony formation assays ( Fig. 2E and 2F) showed that circ-TRPS1 silencing decreased cell proliferation in PC3 and LnCaP cells. Xenograft results validated that circ-TRPS1 knockdown suppressed PC3 tumor growth (measured as volume and weight) comparing with an NC group (Fig. 2G-2I). Immunohistochemical Ki67 detection demonstrated that hsa_circ_0001944 silencing suppressed Ki67 expression in tumor tissues (Fig. 2J), supporting that circ-TRPS1 knockdown suppressed tumor growth. Together, these data suggested that knocking down circ-TRPS1 suppressed the proliferation ability of PCa cells in vivo and in vitro.

circ-TRPS1 downregulation suppressed PCa invasionin vitro and in vivo
Transwell detection showed that circ-TRPS1 knockdown suppressed cell migration in PC3 and LnCaP cells ( Fig. 3A and 3B). The metastasis ability of PC3 cells also decreased after circ-TRPS1 silencing, by live imaging analysis of metastasis model mice (Fig. 3C). Together, these data suggested that knocking down circ-TRPS1 suppressed invasion by PCa cells in vivo and in vitro.
miR-124-3p and EZH2 are downstream targets of circ-TRPS1 in PCa Next, we investigated how circ-TRPS1 regulation affected PCa proliferation and invasion. Previous studies found that circRNA acts as a miRNA sponge. Given that circ-TRPS1 is mainly localized to the cytoplasm, we hypothesized that circ-TRPS1 might regulate tumor biological behavior by acting as a sponge to miRNAs. We employed Interactome to predict miRNAs. The data showed that miR-124-3p might have circ-TRPS1 acting as sponge (Fig. 4A). To validate the association between circ-TRPS1 and miR-124-3p, we prepared wild-type (WT) or mutated (MUT) circ-TRPS1 sequences that included miR-548 binding sequences into a luciferase reporter vector (Fig. 4A). We transfected this reporter vector into 293T cells with or not miR-124-3p mimic. Luciferase reporter analysis suggested that miR-124-3p inhibited luciferase activity in WT-transfected, while not MUT-transfected cells (Fig. 4B), suggesting that miR-124-3p was circ-TRPS1 target.
We conducted tumor sphere formation assays. Upregulating miR-124-3p decreased cancer stem cell differentiation by inhibiting tumor sphere formation. These results indicated that overexpressing EZH2 increased the sphere percentage in LnCaP and PC3 cells (Fig. 6K-MI). Western blots showed that overexpressing EZH2 increased expression of stemness markers OCT4 and Nanog expression in LnCaP and PC3 cells even after upregulating miR-124-3p ( Fig. 6N and 6O). The results suggested that miR-124-3p decreased PCa progression by regulating EZH2-mediated stemness.

Discussion
Increasingly, studies nd that abnormal expression of circRNA is associated with human traits such as cancers [16]. Studies have found that silencing circZNF609 restrains growth, invasion and migration through upregulating microRNA-186-5p in PCa [11]. Upregulated circZMIZ1 enhances PCa cell proliferation and is a valuable plasma marker [17]. In this study, we investigated expression of circRNA using H-PCa and L-PCa tissues and high-throughput sequencing. The results showed that circ-TRPS1 signi cantly increased in H-PCa samples compared with L-PCa. High expression of circ-TRPS1 indicated poor progress, high pathological T stage, lymph node metastasis, and distant metastasis. This result suggested that circ-TRPS1 was involved in the progress of PCa. FISH validated that circ-TRPS1 was distributed mainly in the cytoplasm, similar to other circRNAs [18].
Further study found that circ-TRPS1 expression increased in PCa cell lines. Downregulation of circ-TRPS1 signi cantly decreased invasion and proliferation in in vitro and in vivo, suggesting that circ-TRPS1 had oncogenic characteristics. CircRNAs are reported to interact with miRNAs sponging or stabilizing them [19,20]. The CircInteractome web tool was developed to map RNA-binding protein-and miRNA-binding sites on human circRNA [21]. We used this website for analysis and found that circ-TRPS1 interacted with many miRNAs. The combination with miR-124-3p is the most conservative. Luciferase reporting assays con rmed that miR-124-3p interacted with circ-TRPS1. RT-qPCR showed that silencing circ-TRPS1 promoted miR-124-3p expression, but miR-124-3p overexpression did not reverse circ-TRPS1 lever after circ-TRPS1 silence. This result suggested that miR-124-3p was a circ-TRPS1 downstream target.
Bioinformatic and luciferase reporting experiments con rmed that EZH2 was a direct miR-124-3p target.
Enhancer of Zeste 2 (EZH2) is a gene that is highly conserved as well as expressed in multiple human cancers [27]. EZH2 is aberrantly expressed in glioma and has strong effects on PCa proliferation and metastasis [28][29][30]. Studies show that EZH2 functions in the maintenance of cancer stem-like cell (CSC) properties [31]. CSCs take part in tumor proliferation, recurrence, invasion, and chemoradioresistance, which makes a new concept of tumorigenicity and progression [32]. In this study, we found that circ-TRPS1 silencing suppressed CSC differentiation using tumor sphere formation assays and western blots.

Conclusions
In conclusion, our investigation revealed that circ-TRPS1 was increased in PCa patient samples and cell lines, and upregulation was correlated to aggressive PCa phenotypes. In addition, circ-TRPS1 silencing suppressed PCa metastasis and proliferation in vivo and in vitro. We also discovered that circ-TRPS1 downregulation suppressed PCa migration and proliferation through targeting the miR-124-3p/EZH2 axis.