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Circ-TOP2A acts as a ceRNA for miR-346 and contributes to glioma progression via the modulation of sushi domain-containing 2

Dysregulated circular RNAs (circRNAs) are involved in the carcinogenesis and progression of multiple human malignancies. Knowledge of circRNAs in glioma (GM) is limited and further study to uncover new therapeutic targets for GM is urgently required. The present study demonstrated that circ-TOP2A was elevated in GM tissue specimens and cells and that circ-TOP2A levels indicated an unfavorable clinical prognosis in GM. Functionally, circ-TOP2A knockdown reduced viability, migration and invasion and triggered apoptosis in LN229 cells. Ectopic expression of circ-TOP2A aggravated these malignant behaviors in U87MG cells. In terms of mechanism, RNA-seq was performed to discover the potential targets regulated by circ-TOP2A. Circ-TOP2A acted as a competing endogenous RNA to upregulate sushi domain-containing 2 (SUSD2) expression by sponging microRNA (miR) 346. Rescue assays revealed that the oncogenic function of circ-TOP2A was partially dependent on its regulation of the miR-346/SUSD2 axis. In conclusion, the present study identified that circ-TOP2A promoted GM proliferation and aggressiveness via miR-346/SUSD2 signaling, which is a potential prognostic biomarker and therapeutic target for GM.


Introduction
Glioma (GM) is the commonest type of primary brain tumor (1). Based on the histopathologic classification formulated by the World Health Organization, glioma can be graded into four phases (I-IV) (2). The overall survival rate of GM patients is only ~14 months (3). Although novel therapies against GM have been applied, the situation is still grim for patients (4). At present, the pathogenesis of GM remains elusive.
Circular RNAs (circRNAs) are characterized by a closed-loop structure with limited protein coding capacity (5). The majority of these covalently closed loop circRNAs are produced with exons via the head-to-tail junction (6). Previously, they were believed to be functionless owing to errors in splicing (7). With the development of high-throughput RNA sequencing, they were identified as important factors in numerous biological processes (8). They are relatively stable and resistant to ribonuclease R digestion, making them better biomarkers for diagnosis and treatment than linear RNAs (9). Accumulating evidence suggests that circRNAs can be regulators in the development and progression of cancers (10,11). Nevertheless, the effects and molecular mechanisms of circRNAs in GM are not fully understood and thus need to be extensively and systemically investigated.
Previously, Wang et al (12) performed circRNA sequencing and identified several dysregulated circRNAs in GM tissues relative to their normal counterparts. The present study aimed to identify the potential therapeutic target for GM from the view of circRNAs.

Materials and methods
Clinical specimens. A total of 74 pairs of GM/matched noncancerous specimens (Table SI) were harvested from patients at the Second Affiliated Hospital of Qiqihar Medical University, Heilongjiang, China, between January 2013 and January 2015. A validation cohort consisting of 74 GM patients (age range: 23-71 years old; 49 male, 25 female) was selected according to the following inclusion criteria: i) Patients were diagnosed with GM by preoperative imaging examination (Fig. 1A) and postoperative pathology; ii) patients who underwent radical resection with a clear surgical margin; iii) patients with available follow-up information; iv) patients with a survival time of more than 1 month; v) none of the patients received anticancer treatment before the surgery; and vi) patients who had no history of other malignancies. Exclusion criteria were patients with serious diseases or severe chronic diseases, such as cardiovascular and cerebrovascular diseases. The research was authorized by the Ethics Committee of our hospital (approval number: KY2020-012) and written informed consent was acquired from each patient.
Cell lines and transfection. LN229, U251 and U87MG (cat. no. TCHu138, glioblastoma of unknown origin) were provided by the Chinese Academy of Sciences and normal human astrocytes (NHA; cat. no. CC-2565) were acquired from Lonza Group Ltd. All the cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS, Invitrogen; Thermo Fisher Scientific, Inc.) in a cell incubator (37˚C, 5% CO 2 ). The sequences of circ-TOP2A and SUSD2 were synthesized and cloned into the pcDNA 3.1 circRNA mini vector and pcDNA 3.1 vector, respectively. Short interfering (si)RNAs specific against circ-TOP2A and SUSD2, miR-346 mimics and inhibitors were purchased from GenePharma (Shanghai, China). The plasmids and small RNAs were transfected using Lipofectamine ® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Serum-free medium (125 µl) was used to dilute 5 µl of Lipofectamine ® 3000 in a 1.5 ml EP tube. Meanwhile, 5 µl of siRNA (20 µM) or 2.5 µg of plasmid vector with 5 µl of P3000™ reagent was diluted in 125 µl serum-free medium. After five min of incubation at room temperature, the reagents in the two tubes were combined. After 15-20 min, the mixtures were added into a 2.5-cm dish filled with serum-free medium. Following 8 h of incubation, the medium was replaced with medium containing 10% FBS. Transfection was confirmed by reverse transcription-quantitative (RT-q)PCR at 48 h after transfection. The targeted sequences of the siRNAs specifically targeting circ-TOP2A were: si-circ-TOP2A-1, 5'-ATG CAA CTC TAT GAC ATG GAT-3' and si-circ-TOP2A-2, 5'-CAT GCA ACT CTA TGA CAT GGA-3'.
RT-qPCR. RT-qPCR was performed as previously described (13). Briefly, total RNA was extracted with the TRIzol ® (Thermo Fisher Scientific, Inc.) method according to routine RNA extraction procedures in the laboratory according to the manufacturer's protocol. The isolated RNA was reverse transcribed into cDNA (Roche Diagnostics) in accordance with the manufacturer's instructions. Then, RT-qPCR assay was conducted on a 7500 fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green Master (Roche Diagnostics) according to the manufacturer's instructions. The reaction volume was 50 µl. The thermocycling conditions were as follows: 90˚C for 5 min, 90˚C for 15 sec and 60˚C for 30 sec for 40 cycles. U6 and GAPDH were used as internal controls. PCR primers were as follows: circ-TOP2A, forward, 5'-GGC AGA GAG AGT TGG ACT ACA C-3' and reverse, 5'-CTT CTC CAT TGA AGG GCT TGA G-3'; U6, forward, 5'-ATT GGA ACG ATA CAG AGA AGA TT-3' and reverse, 5'-GGA ACG CTT CAC GAA TTT G-3' and GAPDH, forward 5'-GGG AGC CAA AAG GGT CAT-3' and reverse, 5'-GAG TCC TTC CAC GAT ACC AA-3'. Each reaction was performed in triplicate and results were calculated using the 2 −ΔΔCq method (14).
Actinomycin D treatment. A total of 2.5x10 5 cells were inoculated in 6-well plates and cultured for 48 h at 37˚C. Then, the transcriptional inhibitor actinomycin D (EMD Millipore) was added to the culture medium at 2 mg/ml for 0, 4, 8, 12 and 24 h at 37˚C. Then, total RNA was extracted, and RT-qPCR was used to detect the expression levels of circ-TOP2Aand TOP2A mRNA, as aforementioned.
Western blotting. Proteins were isolated using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was detected using a BCA detection kit (Beijing Solarbio Science & Technology Co., Ltd.). Then, 30 µg samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After immersion in 5% skimmed milk at 22-25˚C for 2 h, the membrane was incubated with anti-SUSD2 (1:2,000; cat. no. ab182147) and anti-GAPDH (1:10,000; cat. no. ab181602; both Abcam) primary antibodies overnight at 4˚C. After incubation with the HRP-conjugated secondary antibody (1:5,000; cat. no. ZB-2306; OriGene Technologies, Inc.) for 2 h at room temperature, the membrane was visualized using an ECL kit (Beyotime Institute of Biotechnology) using a full-automatic chemiluminescence imaging analysis system (Tanon Science and Technology Co., Ltd.). Densitometry was analyzed using ImageJ software (National Institutes of Health).

Subcellular fractionation test.
A PARIS kit (Thermo Fisher Scientific, Inc.) was used to separate RNAs in the cytoplasmic and nuclear fractions. RT-qPCR was performed as aforementioned, with U6 and GAPDH as the nuclear and cytoplasmic controls, respectively.
RNA immunoprecipitation (RIP). RIP was conducted using the Magna RIP RNA-Binding Protein immunoprecipitation kit (EMD Millipore) in accordance with the manufacturer's protocols. After transfection for 48 h, GM cells were lysed with RIP lysis buffer. Afterward, cell lysates were incubated with magnetic beads conjugated with anti-Ago2 (1:50; cat. no. ab186733; Abcam), or anti-IgG at 4˚C for 8 h. The beads were then washed and incubated with Proteinase K to remove the proteins. After purification, the enrichment of circ-TOP2A was tested using RT-qPCR.
RNA pulldown assay. The biotin-labeled circ-TOP2A probe targeting the junction sequence of circ-TOP2A and oligonucleotide probes (500 pmol) were designed and synthesized in vitro by Wuhan GeneCreate Biological Engineering Co., Ltd. and used for incubation with cell lysates at 4˚C overnight. Then, the complex was incubated with streptavidin-conjugated magnetic beads (Invitrogen; Thermo Fisher Scientific, Inc.) at 22-25˚C for 2 h. After purification and RNA extraction (TRIzol method) (15), the enrichment of circ-TOP2A and miRs was measured using RT-qPCR.
293T cells were co-transfected with miR-346 mimics and plasmids containing the 3'-UTRs of wild-type or mutant sequences of the miR binding site in SUSD2 or wild-type or mutant sequences of circ-TOP2A using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. After transfection for 36 h, the relative luciferase signals of the cells were measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). The specific target activity was expressed as the relative activity ratio of firefly luciferase to Renilla luciferase. The experiment was repeated three times independently.
Cell Counting . Cell viability was assessed using the CCK-8 assay (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. In brief, the cells were collected and the concentration was adjusted to 2x10 3 cells/well before they were maintained in a cell incubator for 0, 24, 48, 72 and 96 h. The cells were then seeded in 96-well plates at 1,500 cells per well. After the indicated specific treatments, 10 µl of CCK-8 was supplied to the wells and maintained at 37˚C for 2 h. Then, the absorbance of each well at 450 nm was tested using a microplate reader (Thermo Fisher Scientific, Inc.).
Colony forming test. LN229 and U87MG cells were plated into 6-well plates (1,000 cells/well) with medium containing 10% FBS and incubated at 37˚C. After ~10 days, the colonies were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with crystal violet solution (Sigma-Aldrich; Merck KGaA) for another 20 min at room temperature. Finally, images were captured of the colonies and they were counted manually.
Apoptosis detection. Cell apoptosis assays were performed by flow cytometry (Applied Biosystems; Thermo Fisher Scientific, Inc.). First, GM cells were seeded in 6-well plates before collection by centrifuging at 1,000 x g for 3 min at room temperature. The cells were washed twice with precooled PBS and the concentration was adjusted to 5x10 5 -5x10 6 cells with 400 µl of staining buffer (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl and 2.5 mM CaCl 2 ) containing 5 µl of Annexin V labeled with fluorescein isothiocyanate and 5 µl of PI (Annexin V-FITC apoptosis kit; BD Biosciences). Following a 20-minute incubation at 22-25˚C in the dark, the treated cells were subjected to apoptosis assays by flow cytometry (FACScan; BD Biosciences). FlowJo v10 software (Tree Star, Inc.) was used for apoptosis analysis. The percentage of early + late apoptotic cells was calculated as the apoptotic rate.
Transwell experiments. A Transwell plate containing an 8-µm pore size filter (BD Biosciences) was used to detect the invasion and migration of cells. The cells were suspended in RPMI-1640 with 0.1% FBS and supplied to the top compartment at a density of 10,000 cells per well. For the cell invasion assay, Matrigel was pre-cooled at 4˚C overnight and coated on the upper side of the membrane. Then, the chambers were placed in a 24-well plate and 600 µl of medium containing 10% FBS was added to each well. After incubation in a cell incubator at 37˚C for 24 h, the cells on the upper side of the membrane were removed with cotton swabs and the migrated/invaded cells in the lower membrane of the chambers were fixed by paraformaldehyde at room temperature for 20 min and then stained with crystal violet for 20 min at room temperature. Images were captured from five randomly selected fields of view (magnification, x200) under a light microscope.
Data analysis. All results are presented as the mean ± standard deviation and were analyzed with GraphPad Prism 8.0 (GraphPad Software, Inc.) and SPSS 22.0 (IBM Corp.). Unpaired Student's t test was used to compare the significance of differences between two groups. For comparisons between cancerous and adjacent normal tissues, paired t test was used. One-way ANOVA with post hoc Tukey's test was used to compare the significance of differences among three or more groups. Kaplan-Meier analysis with log-rank test was applied to measure the overall survival rate (from surgery to death) and progression-free survival (from surgery to tumor recurrence/metastasis) of GM patients. The Cancer Genome Atlas (TCGA) and Pearson's correlation analysis was used to assess the correlation between SUSD2 and miR-346 expression levels, as well as patient survival data. P<0.05 was considered to indicate a statistically significant difference.

Results
Circ-TOP2A is overexpressed in GM and is associated with poor prognosis. A total of five circRNAs, circ-CLSPN, circ-TOP2A, circ-MELK, circ-BUB1 and circ-KIF4A, were selected from the sequencing data (12). Total RNA was isolated from 10 pairs of GM and normal tissues to detect the expression of circRNAs. As shown in Fig. 1B, circ-TOP2A was the most upregulated circRNA expressed in GM tissues compared with its the normal counterparts. Therefore, it was chosen for further study. Circ-TOP2A (circ_0043548) was mapped to chr17:38556460-38569223. It was spliced from exons 7-23 of TOP2A. The spliced variant of circ-TOP2A was 2424 nucleotides long (Fig. 1C). It was determined that circ-TOP2A was more stable than TOP2A mRNA (Fig. 1D). In addition, total RNA was extracted to detect the expression of circ-TOP2A and linear TOP2A mRNA following treatment with actinomycin D at different time points. Linear TOP2A showed a shorter half-life compared with circ-TOP2A (Fig. 1E). RT-qPCR demonstrated that circ-TOP2A was markedly elevated in GM specimens compared with noncancerous samples (Fig. 1F). To explore the prognostic value of circ-TOP2A, patients were divided into high-and low-circ-TOP2A groups according to the median expression level of circ-TOP2A in cancer tissues. As illustrated in Fig. 1G and H, Kaplan-Meier curves revealed that overall survival rate (P=3.1x10 -2 ) and progression-free survival (P=3.5x10 -2 ) were lower in the high-circ-TOP2A group compared with the low-circ-TOP2A group. Furthermore, the expression of circ-TOP2A was found to be overexpressed in GM cell lines compared with NHA cells (Fig. 1I).
Circ-TOP2A promotes GM cell proliferation and aggressiveness. The cellular function of circ-TOP2A by down-/up-regulation of circ-TOP2A was then examined in GM cell lines. LN229 cells were used for the knockdown study due to their high expression of circ-TOP2A. The silencing efficiencies of si-circ-TOP2A-1 and si-circ-TOP2A-2 were favorable ( Fig. 2A). Circ-TOP2A expression was ectopically expressed in U87MG cells due to its lowest level of circ-TOP2A among the enrolled GM cell lines ( Fig. 2A). As presented in Fig. 2B, transfection with circ-TOP2A siRNAs or vector could not down-/up-regulate TOP2A mRNA expression levels. Functionally, silencing of circ-TOP2A significantly impeded cell viability and clone-forming ability in LN229 cells ( Fig. 2C and E). Overexpression of circ-TOP2A caused the opposite effect in U87MG cells (Fig. 2D and F). Additionally, down and upregulation of circ-TOP2A respectively triggered or inhibited cell apoptosis in GM cells (Fig. 2G and H). In addition, circ-TOP2A downregulation suppressed cell migration and invasion in LN229 cells (Fig. 2I). In contrast, overexpression of circ-TOP2A exerted the opposite effect in U87MG cells (Fig. 2J).
Circ-TOP2A/miR-346/SUSD2 signaling is critical for GM cell function. Rescue experiments were performed to reveal the mechanisms of circ-TOP2A in GM. As shown in Fig. 5A, the protein level of SUSD2 was decreased in the si-circ-TOP2A-1 group and changes in SUSD2 expression were reversed by co-transfection with miR-346 inhibitor or SUSD2 vector. In addition, SUSD2 expression was elevated following transfection with the circ-TOP2A-overexpressing vector in U87MG cells. After co-transfection with miR-346 mimics or si-SUSD2, the protein expression of SUSD2 was partly inhibited (Fig. 5A). Additionally, relative expression of circ-TOP2A was decreased following transfection with si-circ-TOP2A-1. Si-circ-TOP2A-1 co-transfection with miR-346 inhibitor or SUSD2 vector did not alter circ-TOP2A expression in LN229 cells. Similarly, circ-TOP2A vector led to increased expression of circ-TOP2A. Further co-transfection with miR-346 mimics or si-SUSD2 had no effect on circ-TOP2A expression in U87MG cells (Fig. S1A). miR-346 expression was unchanged after knockdown or overexpression of circ-TOP2A, which implied that circ-TOP2A sponges miR-346 to inhibit its functions rather than expression levels. Co-transfection with miR-346 inhibitor and miR-346 mimics markedly decreased and increased miR-346 expression, respectively (Fig. S1B). CCK-8, colony formation and Transwell assays indicated that the inhibitory effects of si-circ-TOP2A on cell proliferation and invasion were attenuated by co-transfection with miR-346 inhibitor or SUSD2 vector (Fig. 5B, D and F). Overexpression of the circ-TOP2A vector led to increased cell viability, clone-forming ability and invasive potential in U87MG cells. These malignant behaviors were partially rescued after co-transfection with miR-346 mimics or si-SUSD2 (Fig. 5C, E and G). Taken together, these findings revealed that the circ-TOP2A mediated miR-346/SUSD2 axis triggered GM carcinogenesis.

Discussion
Tumorigenesis and metastasis are complicated processes involving the activation of oncogenes and the inactivation of tumor suppressor genes. CircRNAs are reported to regulate gene expression at epigenetic, transcriptional and posttranscriptional levels, mediating the initiation and metastasis of tumors (10,18,19). Circ-TOP2A was screened as an upregulated circRNA in GM tissue samples compared with noncancerous tissues (12). The current study further identified that the expression of circ-TOP2A was upregulated in GM tissues and cell  Functional experiments indicated the oncogenic role of circ-TOP2A in mediating GM cell growth, migration and invasiveness. RNA-Seq of circ-TOP2A-depleted LN229 and U87MG cells revealed SUSD2 as a target of circ-TOP2A. In addition to interacting with proteins, circRNAs can regulate target gene expression indirectly via competitive binding with miRs (20). For instance, circ-CSPP1 acts as a competing endogenous RNA (ceRNA) to contribute to colorectal carcinoma cell epithelial-mesenchymal transition and liver metastasis by upregulating collagen, type I, α 1 (21). The localization of circRNAs suggests how they exert their functions. Circ-TOP2A was dominantly distributed in the cytoplasm, which suggested that its mechanism functions at the post-transcriptional level. miR-346 was further demonstrated The protein level of SUSD2 was detected by western blotting following transfection in LN229 and U87MG cells. CCK-8 assay was conducted to evaluate the viability of (B) LN229 and (C) U87MG cells following transfection. Colony formation assay was conducted to evaluate the clone forming ability of (D) LN229 and (E) U87MG cells following transfection. Transwell assay was conducted to evaluate the invasion of (F) LN229 and (G) U87MG cells following transfection. B-G: The data are shown as the mean ± standard deviation (n=3). * P<0.05, ** P<0.01. Circ, circRNA; GM, glioma; miR, microRNA; si, short interfering; NC, negative control; inh, inhibitor. as a novel target of circ-TOP2A. miR-346 is reported to target and inhibit the expression of a number of classical oncogenes and then suppress the progression of a number of cancers (22,23). Previously, miR-346 was reported as a tumor suppressor in GM by directly targeting NFIB (24). The present study validated the tumor suppressive function of miR-346 in GM and found that a miR-346 inhibitor could reverse the anticancer effects of circ-TOP2A knockdown. These results demonstrated that circ-TOP2A facilitates GM growth and aggressiveness, at least in part via repression of the function of miR-346. SUSD2, a type I transmembrane protein that localizes to the cell surface, has been reported to be upregulated in a number of tumors and to promote cancer progression and metastasis (25,26). However, some studies have indicated that upregulated SUSD2 impedes cell progression in several malignancies, suggesting a possible tumor suppressive role of SUSD2 (27,28). These previous studies indicate that SUSD2 might have a complex function in different malignancies. Consistent with the studies on breast cancer and ovarian cancer (25,26), SUSD2 was obviously increased in GM tissues and cells compared with matched control groups. Notably, the present study also demonstrated that overexpression of SUSD2 acted as a tumorigenic function in circ-TOP2A-downregulated GM cells, suggesting the cancerogenic function of SUSD2 in GM. Collectively, circ-TOP2A promoted GM proliferation and aggressiveness through the miR-346/SUSD2 signaling pathway.
In conclusion, the results of the present study suggested that circ-TOP2A was increased, circular and stable in GM cells. Notably, upregulation of circ-TOP2A exerted its functions by facilitating cell growth, migration and invasion, while inhibiting apoptosis in GM cells by targeting miR-346/SUSD2 signaling. Therefore, circ-TOP2A may be a potential prognostic biomarker/therapeutic target for patients with GM.