circ_0082375 promotes the progression of glioma by regulating Wnt7B

Abstract Circular RNAs contribute to the progression of glioma. However, the biological role and underlying mechanism of circ_0082375 in glioma remain unclear. Quantitative real-time PCR and Western blot assay were used to evaluate the expression levels of circ_0082375, microRNA-485-5p, and Wnt family member 7B (Wnt7B). The overall survival of glioma patients was estimated by the Kaplan–Meier curve. Cell proliferation, apoptosis, invasion, and migration were detected by cell counting kit-8, 5-ethynyl-2 -deoxyuridine (EdU) staining, flow cytometry, and transwell assays, respectively. Glucose level and lactate production were determined using glucose and lactate assay kits. In vitro angiogenesis assay was used to evaluate the angiogenesis of glioma cells. The interaction between microRNA (miR)-485-5p and circ_0082375 or Wnt family member 7B (Wnt7B) was verified by dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft model was used to verify the function of circ_0082375 in vivo. circ_0082375 was upregulated in glioma tissues, and it was closely related to the prognosis of glioma patients. circ_0082375 knockdown suppressed cell proliferation, migration, invasion, angiogenesis, glycolysis, and epithelial-mesenchymal transition (EMT), and promoted cell apoptosis in glioma cells. irc_0082375 was a sponge of miR-485-5p, which directly targeted Wnt7B. Knockdown of circ_0082375 inhibited the malignancy, angiogenesis, and glycolysis of glioma cells in vitro by sponging miR-485-5p. Besides, circ_0082375 knockdown hampered the growth of glioma growth by regulating the miR-485-5p/Wnt7B axis in vivo. Altogether, circ_0082375 regulated miR-485-5p/Wnt7B axis to promote the malignancy, angiogenesis, and glycolysis of glioma cells, thereby contributing to the progression of glioma.


Introduction
Glioma is a common primary intracranial tumor with strong invasiveness [1]. It was reported that the morbidity of glioma has risen from 5.9 per 100,000 people in 1973 to 6.61 per 100,000 people in 2016 [2]. Although significant progress has been made in surgical and chemotherapy techniques, glioma remains one of most frequent causes of cancer deaths globally [3]. Thus, seeking a novel biomarker for the treatment of glioma patients is urgently required.
MiRNAs are partly responsible for the progression and tumorigenesis of glioma cells [10]. Several miRNAs, such as miR-504 [11], miR-150 [12], and miR-1265 [13], have been proved to be unconventionally expressed in glioma and are implicated in glioma progression by affecting cell growth, EMT, and glycolysis. MiR-485-5p has lower levels in a variety of cancers [14], and it plays an antitumor role in oral squamous cell carcinoma [15] and esophageal cancer [16]. This study validates the underlying mechanism of miR-485-5p in glioma cells.
WNT signaling is one of the most vital pathways for tumor development [17]. Among these genes, myeloid Wnt7B exerts a promoting effect on cell invasion, angiogenesis, and lung metastasis in breast cancer [18]. Importantly, Wnt7B regulates cell proliferation and invasion in glioma [19]. Nevertheless, the effects of Wnt7B on glioma cell malignancy, angiogenesis, and glycolysis need to be further studied.
Here, we aimed to investigate the function of circ_0082375 in glioma. Besides, the impact of the circ_0082375/miR-485-5p/Wnt7B axis on glioma progression was enquired, providing an effective molecular target for glioma therapy.

Tissue specimens and cell
Tumor specimens were obtained from 48 glioma patients who underwent surgery at Qilu Hospital of Shandong University. Normal brain tissues were acquired from 48 patients without glioma who experienced partial brain resection. None of the participants received treatment before surgery.
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration and has been approved by the Ethics Committee of Qilu Hospital of Shandong University.

RNA isolation and RNase R treatment
Total RNA was extracted from glioma tissues and cells using TRIzol reagent (Invitrogen). Cytoplasmic and nuclear RNA purification kit (Amyjet Scientific, Wuhan, China) was used to isolate RNA from the cytoplasmic and nuclear RNA fraction following the manufacturer's instructions.

RT-qPCR
The cDNA was extracted by using PrimeScript TM RT reagent kit (TaKaRa, Wuhan, China) or miScript II RT Kit (TaKaRa). qPCR was performed using a SYBR premix Ex TaqII kit (TaKaRa) on 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used for RT-qPCR were shown in Table 2. GAPDH and U6 were used as the internal controls.

Flow cytometry assay
The transfected U-251 and LN-229 cells were plated in a sixwell plate for 48 h. Then cells were incubated with Annexin V fluorescein isothiocynate and propidium iodide for 20 min based on the manufacturer's instructions. Cell apoptosis was analyzed on a BD-FACS Canto II (BD, San Jose, CA, USA).

Glucose level and lactate production detection
U-251 and LN-229 cells were transfected and further incubated for 48 h. The supernatant of the cell medium was then collected, and the levels of glucose and lactate were estimated by a glucose and lactate assay kit (Sigma-Aldrich). Lactate production was calculated by subtracting the concentration of lactate in the primordial medium from the concentration of lactate in the collected supernatant.

RNA immunoprecipitation (RIP) assay
RIP assay was performed by using a Magna RIP™ RNAbinding protein kit (Millipore, Billerica, MA, USA). U-251 and LN-229 cells were lysed, and the collected supernatants were incubated with magnetic beads precoated with Ago2 antibody or IgG antibody overnight at 4°C. Then RNAs were purified and extracted by TRIzol (Invitrogen), followed by the estimation of the abundance of circ_0082375 or Wnt7B and miR-485-5p using RT-qPCR analysis.

Xenograft model
The animal experiments were executed with the approval of the Animal Research Committee of Qilu Hospital of Shandong University. U-251 cells with stably expressing sh-circ_0082375 or sh-NC were injected into 6-week-old male nude mice (n = 5 per group), which were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Tumor volume was measured once a week. Five weeks after injection, all mice were euthanized using pentobarbital sodium, and tumor tissues were gathered and weighed. In addition, tumor tissues were stored in liquid nitrogen for further examination.

Statistical analysis
GraphPad Prism V6.0 (GraphPad Software, Inc, San Diego, CA, USA) was used to perform the statistical analysis. Data were represented as the mean ± standard deviation (SD). For each expression analysis, three biological replicates were performed. All results were inspected using Student's t-test (two groups) or one-way ANOVA (multiple groups). P < 0.05 was regarded as statistically significant.

circ_0082375 is elevated in glioma tissues and cells
hsa_circ_0082375 (chr7: 129950626-129964020), termed circCPA4, is derived from circularization of exon8, exon9, and exon10 of the CPA4 gene ( Figure 1a). Figure 1b displayed that circ_0082375 was aberrantly elevated in glioma tissues (n = 48) in contrast to 48 cases of normal tissues. Then the 48 cases of glioma patients were divided into high circ_0082375 group (n = 24) and low circ_0082375 group (n = 24) based on the median value of circ_0082375 expression ( Figure 1c). And Kaplan-Meier curves uncovered that patients with high circ_0082375 expression group had worse overall survival (P = 0.0095) than those glioma patients with low circ_0082375 expression ( Figure 1d). Besides, circ_0082375 was preferentially distributed in in the cytoplasm of glioma cells (Figure 1e), indicating that circ_0082375 might act as a miRNA sponge in the cytoplasm. Furthermore, RNase R treatment followed by RT-qPCR assay indicated that circ_0082375, but not its linear transcript CPA4, was resistant to RNase R digestion (Figure 1f). The above results uncovered that circ_0082375 was inextricably linked to the prognosis of glioma patients.

circ_0082375 knockdown induces apoptosis and impedes proliferation, migration, invasion, angiogenesis, glycolysis, and EMT in glioma cells
For investigation of circ_0082375 function in glioma, siRNA against circ_0082375 were transfected into U-251 and LN-229 cells. As displayed in Figure 2a, circ_0082375 was downregulated in glioma cells after si-circ_0082375#1, si-circ_0082375#2, or si-circ_0082375#3 transfection. Si-circ_0082375#1 was used for further investigation due to its high knockdown efficiency. Inhibition of circ_0082375 hampered the proliferation of U-251 and LN-229 cells, as the OD value ( Figure 2b) and EdU-positive cells (red; Figure 2c) are decreased in cells with circ_0082375 knockdown, in contrast with the si-NC group. Flow cytometry assay uncovered that knockdown of circ_0082375 elevated the apoptosis rate of glioma cells (Figure 2d). Besides, cell migration and invasion were also curbed in cells with circ_0082375 knockdown (Figure 2e and f). Furthermore, circ_0082375 knockdown decreased the angiogenesis of HUVEC cells cocultured with glioma cells (Figure 2g). In addition, the elevated levels of glucose in the culture medium ( Figure 2h) and the decreased levels of lactate production (Figure 2i) in cells with circ_0082375 knockdown, suggesting that the glycolysis of glioma cells was inhibited by circ_0082375 knockdown. Furthermore, the protein levels of proliferative marker PCNA, apoptosis marker cleaved-caspase 3, and EMT markers containing E-cadherin, N-cadherin, FN, and snail were detected. As shown in Figure 2j and k, PCNA protein levels were elevated while the ratio of cleaved-caspase 3/total-caspase 3 was elevated in glioma cells with circ_0082375 knockdown. Besides, circ_0082375 knockdown inhibited EMT phenotypes in glioma cells, with the upregulation of E-cadherin and the downregulation of N-cadherin, FN, and snail (Figure 2j and k). These results uncovered that circ_0082375 promotes glioma cell progression by regulating proliferation, apoptosis, migration, invasion, angiogenesis, glycolysis, and EMT.

circ_0082375 functions as a molecule sponge for miR-485-5p
CircRNAs are reported to perform their biological functions by serving as competing endogenous RNAs or endogenous sponge for miRNAs [21]. Bioinformatics software Starbase v2.0 (http://starbase.sysu.edu.cn/) database showed that miR-485-5p might interact with circ_0082375 (Figure 3a). For the sake of confirmation, we constructed the wildtype (circ_0082375-wt) and mutant-type (circ_0082375-mut) reporter plasmids of circ_0082375. Figure 3b disclosed  or si-NC were determined by Western blot. All data were presented as the mean ± SD from three independent experiments. Statistical significance was determined using Student's t test. *P < 0.05.
that transfection of miR-485-5p mimic obviously decreased the luciferase activity of the circ_0082375-wt group rather than the circ_0082375-mut group, compared with the cells with NC transfection. Besides, circ_0082375 and miR-485-5p were significantly gathered in cells incubated with anti-Ago2 antibody in contrast with cells incubated with anti-IgG antibody (Figure 3c). Thus, we confirmed that circ_0082375 served as a miR-485-5p sponge. Furthermore, the levels of miR-485-5p in U-251 and LN-229 cells were downregulated by circ_0082375 overexpression and upregulated by circ_0082375 silencing (Figure 3d). In addition, miR-485-5p expression in 48 cases of glioma tissues was noticeably decreased compared to normal tissues (n = 48; Figure 3e), and a negative correlation (r = −0.7531, P < 0.0001) was found between circ_0082375 and miR-485-5p expression in 48 cases of glioma tissues (Figure 3f). Altogether, circ_0082375 acted as a molecule sponge for miR-485-5p.

Knockdown of miR-485-5p rescues circ_0082375 silencing-mediated suppression effects on the progression of glioma cells
Rescue experiments were performed, and the results exhibited that miR-485-5p levels were upregulated in cells with si-circ_0082375#1 transfection, whereas this elevation was restored by cotransfection of anti-miR-485-5p (Figure 4a). Besides, miR-485-5p knockdown partly reversed the suppressive effect of si-circ_0082375#1 transfection on cell proliferation (Figure 4b and c). The promoting effect of si-circ_0082375#1 transfection on cell apoptosis (Figure 4d) and the inhibiting effects of circ_0082375 silencing on cell migration (Figure 4e), invasion (Figure 4f), angiogenesis (Figure 4g), as well as glycolysis (Figure 4h and i), were  (j and k) Western blot assay was used to measure the protein levels of PCNA, cleaved-caspase 3, total-caspase 3, N-cadherin, E-cadherin, FN, and snail. All data were presented as the mean ± SD from three independent experiments. Statistical significance was determined using the one-way ANOVA method. *P < 0.05.
partly reversed by downregulation of miR-485-5p. And it was further confirmed that the inhibitory effects of circ_0082375 knockdown on PCNA, N-cadherin, FN, and snail protein levels, as well as the promoting effects on E-cadherin protein levels and the ratio of cleaved-caspase 3/total-caspase 3 were partly rescued by miR-485-5p inhibitor (Figure 4j and k). Thus, these findings show that circ_0082375 promotes glioma progression by negatively regulating miR-485-5p expression.

circ_0082375 promotes Wnt7B expression by interacting with miR-485-5p
Subsequently, the downstream targets of miR-485-5p were predicted by the Starbasev2.0 database and found that Wnt7B 3'-UTR sequence harbored complementary binding sites to miR-485-5p (Figure 5a). Besides, cotransfection of miR-485-5p mimic and Wnt7B-wt restrained the luciferase activity more than half in contrast with the NC group. However, miR-485-5p mimic did not significantly affect the luciferase activity of the Wnt7B-mut group (Figure 5b). As expected, both miR-485-5p and Wnt7B were gathered by the anti-Ago2 antibody (Figure 5c). Furthermore, we observed that circ_0082375 overexpression rescued the inhibiting effect of miR-485-5p on Wnt7B protein levels in U-251 and LN-229 cells (Figure 5d). Moreover, Wnt7B mRNA and protein levels were prominently elevated in 48 cases of glioma tissues (Figure 5e and f). Furthermore, Wnt7B expression was positively correlated with circ_0082375 expression (P < 0.0001, r = 0.5773) in glioma tissues, whereas it negatively correlated with miR-485-5p expression (P < 0.0001, r = −0.7087; Figure 5g and h).  (g and h) The correlation between Wnt7B and circ_0082375 or miR-485-5p in glioma tissues was evaluated by the Pearson correlation coefficient. All data were presented as the mean ± SD from three independent experiments. Statistical significance was determined using the one-way ANOVA method. *P < 0.05.  (Figure 6j and k). These findings uncovered that miR-485-5p restrains glioma development by suppressing Wnt7B expression.

circ_0082375 deficiency curbs the growth of glioma tumor in vivo
For investigation of the function of circ_0082375 in vivo, xenograft tumor models were established by injecting U-251 cells stable expressing sh-NC or sh-circ_0082375 (five mice in each group). In contrast with the sh-NC group, the tumor volume and tumor weight were declined in the sh-circ_0082375 group (Figure 7a and b). Besides, RT-qPCR assay revealed that circ_0082375 was downregulated, whereas the miR-485-5p level was upregulated ( Figure 7c) in tumor tissues derived from nude mice injected with U-251 cells carrying sh-irc_0082375. Furthermore, the protein levels of Wnt7B and PCNA were declined, whereas the ratio of cleaved-caspase 3/total-caspase 3 was (e) Immunohistochemistry analysis of Ki-67 in tissue sections in sh-circ_0082375 and sh-NC groups. All data were presented as the mean ± SD from three independent experiments. Statistical significance was determined using Student's t test. *P < 0.05. elevated (Figure 7d) in tumor tissues from the sh-irc_0082375 group. Furthermore, immunohistochemistry with anti-ki-67 antibody showed that circ_0082375 knockdown markedly reduced the proliferation of glioma cells (Figure 7e). These results demonstrated that interference of circ_0082375 efficiently impedes the growth of glioma in vivo.

Discussion
Mounting evidence suggests that circRNAs are partly responsible for the tumorigenesis and progression of glioma. circ_0082375 (hsa_circCPA4_007), derived from the CPA4 gene, was first identified to be significantly increased in glioma by high-throughput circRNA microarray assay. And circ_0082374 (hsa_circCPA4_006) has been reported to accelerate cell proliferation and metastasis by adsorbing miRNA let-7 [9]. In our research, elevated circ_0082375 expression was found in 48 cases of glioma tissues, and the outcome of glioma patients was closely related to the expression level of circ_0082375. Unlike normal cells that rely on mitochondrial oxidation to produce energy, tumor cells mainly generate metabolic energy by glycolysis [22]. In addition, the intermediates produced during glycolysis contribute to the growth of tumor cells [23]. Besides, angiogenesis exerts a pivotal role in glioma, as new blood vessels are indispensable for the survival and progress of glioma and can transport the necessary oxygen, growth factors, and nutrients [24]. Furthermore, research have disclosed that angiogenesis exhibits a closely intertwined with the outcome of glioma patients [25,26]. In our research, circ_0082375 knockdown inhibited cell proliferation, migration, invasion, angiogenesis, EMT, and impaired cell glycolysis and promoted cell apoptosis in glioma cells. Altogether, circ_0082375 served as a tumor promoter in glioma in vitro.
Emerging evidence reveals that some circRNAs located in the cytoplasm can interact with miRNAs through miRNA response elements [27]. In this study, circ_0082375 was mainly located in the cytoplasm of glioma cells, suggesting that circ_0082375 might function as a miRNA sponge. Through luciferase assay and RIP assay proved that circ_0082375 acted as a miR-485-5p sponge. A previous study revealed that MiR-485-5p curbed cell proliferation and motility in glioma [28]. In accordance with the previous research, miR-485-5p was downregulated in glioma tumors. Furthermore, repression of miR-485-5p could abolish the inhibiting impacts of circ_0082375 silencing on cell progression, migration, invasion, angiogenesis, EMT, and glycolysis, indicating the tumor-suppressive function of miR-485-5p in glioma.
Subsequently, the downstream targets of miR-485-5p were investigated, and we authenticated that Wnt7B acted as a target of miR-485-5p. As a member of the WNT signaling pathway, Wnt7B has been found to regulate the growth of mammalian cells [29]. In accordance with previous research, Wnt7B was highly expressed in glioma tissues and cells [30,31]. Our findings disclosed that the suppressive effects of miR-485-5p mimic on cell progression, migration, invasion, angiogenesis, EMT, and glycolysis could be counteracted by overexpression of Wnt7B. Besides, the authors found that circ_0082375 upregulated Wnt7B expression by sponging miR-485-5p in glioma cells. Consistent with in vitro results, circ_0082375 knockdown hampered tumor growth by regulating the miR-485-5p/Wnt7B axis.
The novelty of this study was the first report of the molecular mechanism of the circ_0082375/miR-485-5p/ Wnt7B axis in glioma. Unfortunately, we did not explore whether circ_0082375 can be used as a diagnostic biomarker. In addition, the function of circ_0082375 overexpression in glioma in xenograft models can be explored in the future.
Together, our results demonstrated that circ_0082375 increased Wnt7B expression via sponging miR-485-5p to regulate cell proliferation, apoptosis, migration, invasion, angiogenesis, glycolysis, and EMT, thereby affecting glioma progression. These findings strengthened our comprehension on the progression of glioma and provided a novel target for treatment of the glioma.