Knockdown of circ_CLIP2 regulates the proliferation, metastasis and apoptosis of glioma cells through miR-641/EPHA3/STAT3 axis

Abstract A great amount of reaches have confirmed that circular RNAs (circRNAs) are novel regulators in glioma progression. Here, our work aimed to probe the specific role of circ_CLIP2 in glioma. The mRNA and protein expressions were analyzed by qRT-PCR and western blot, respectively. Cell viability, migration, invasion and apoptosis were examined by MTT assay, tranwell and flow cytometry assays, respectively. Moreover, the binding relationships between circ_CLIP2, microRNA (miR)-641 and erythropoietin-producing human hepatocellular (Eph)A3 were verified by dual luciferase reporter gene assay and/or RIP assay. The following data showed that circ_CLIP2 and EPHA3 were markedly increased in glioma tissues and cells, while miR-647 was downregulated. Gain- and loss-of-function experiments discovered that circ_CLIP2 knockdown remarkably inhibited cell proliferation, migration and invasion and promoted cell apoptosis of glioma cells, while these effects of circ_CLIP2 knockdown were abolished by miR-641 inhibition. Circ_CLIP2 was proved as a sponge of miR-641 to competitively upregulate EPHA3 expression. In addition, EPHA3 overexpression could abolish the inhibitory effects of miR-641 overexpression on the malignant behaviors of glioma cells by activating the signal transducer and activator of transcription 3 (STAT3). These findings elucidated that circ_CLIP2 knockdown suppressed glioma development by regulation of the miR-641/EP HA3/STAT3 axis, which provided a novel mechanism for understanding the pathogenesis of glioma.


Introduction
Glioma is the most common primary central nervous system tumor with a poor prognosis (Mamelak & Jacoby, 2007;Zhou et al., 2020), accounting for more than 50% of all of the intracranial tumors (Yi et al., 2013).The current treatment methods for glioma mainly include surgery, chemotherapy and radiotherapy (Yamaguchi, 2017).However, the overall therapeutic effects are still unsatisfactory due to the unclear pathogenesis and lack of specific therapeutic targets.Recently, gene therapy for glioma has been widely studied, but the specific target and mechanism have not yet been well determined (Kim et al., 2018).Hence, exploring the new molecular mechanism of glioma is of great value for its treatment and prognosis.
Circular RNAs (CircRNAs) are a class of non-coding RNA molecules with a loop structure (Memczak et al., 2013).The accumulating evidence have displayed that circRNAs are involved in regulating glioma progression (Jiang et al., 2020;Sun et al., 2020;Yang et al., 2018).As proof, circ_PTN overexpression could promote glioma growth in vivo and in vitro (Chen et al., 2019).Circ_ PIP5K1A was upregulated in glioma tissues, and its overexpression facilitated glioma cell proliferation and invasion (Zheng et al., 2021).In addition, circ_0014359 knockdown remarkably repressed glioma cell malignant behaviors (Shi et al., 2019).Circ_CLIP2 is a newly discovered cancerrelated circRNA, which is a powerful regulator in tumors.Notably, circ_CLIP2 is confirmed to act as an oncogenic role in glioma by several studies (Song et al., 2016;Xiao et al., 2021).Song et al. demonstrated that circ_CLIP2 was markedly upregulated in glioma tissues (Song et al., 2016).In addition, circ_CLIP2 knockdown repressed glioma progression via targeting miR-195-5p/HMGB3 signaling (Xiao et al., 2021).These observations suggested circ_CLIP2 was a risk factor in glioma progression, while the specific mechanism of circ_CLIP2 in glioma remained unclear.
It is well known that circRNAs function as competing endogenous RNAs (ceRNAs) by sponging microRNAs (miRNAs), in turn affecting the binding of miRNAs to their targets, which is described as a novel RNA regulation mechanism (Song et al., 2020).In the current study, we found that circ_CLIP2 had a binding site to miR-641 by using StarBase prediction.Recently, miR-641 was widely functioned as a tumor suppressor in many types of human malignancies (Kong et al., 2018;Wang et al., 2019;Zhang et al., 2020).For instance, miR-641 overexpression could remarkably repress lung cancer cell malignant behaviors (Kong et al., 2018).Additionally, miR-641 overexpression resulted in cell cycle arrest of gastric cancer cells through targeting MDM2 (Wang et al., 2019).Notably, it was observed that miR-641 was upregulated in glioma, and miR-641 inhibitor transfection remarkably facilitated glioma cell migration and invasion via forming a COX10-AS1/miR-641/E2F6 feedback loop (Liu et al., 2021).However, whether circ_CLIP2 affects glioma progression by sponging miR-641 remains elusive.
The erythropoietin-producing human hepatocellular (Eph) receptors refer to transmembrane glycoprotein members of the tyrosine kinase receptors family (Janes et al., 2014).EPHA3, as one receptor of the 14 different Ephs, is involved in regulating biological processes, including angiogenesis, stem cell maintenance and metastasis, etc (Janes et al., 2014).Generally, EPHA3 usually served as an oncogene in multiple human cancers (London & Gallo, 2020).EPHA3 upregulation was associated with poor prognosis of cancer patients (London & Gallo, 2020).More importantly, it has been confirmed that EPHA3 has the potential to be a functional tumor-specific therapeutic target in glioma (Day et al., 2013).Specifically, EPHA3 is markedly upregulated in recurrent glioma and functions in maintaining self-renewal and tumorigenesis (Offenh€ auser et al., 2018).Moreover, the inhibition of EPHA3 could significantly inhibit the disease progression of glioma patients and improve the overall survival rate of patients (Offenh€ auser et al., 2018).As previously reported, EPHA3 regulated the development of gastric cancer through STAT3 signaling (Lv et al., 2018).STAT3, as a member of the STAT family, functions in transmitting signals from cytokines and growth factors.It's observed that STAT3 is activated in many types of human malignancies including glioma (Luwor et al., 2013;Mukthavaram et al., 2015).Therefore, it's suggested that the EPHA3/STAT3 signaling pathway may act as a vital role in promoting glioma progression.Nevertheless, the role of the EPHA3/STAT3 signaling pathway in glioma needs further investigation.
Herein, we aimed to probe the function and its molecular mechanism of circ_CLIP2 in glioma.In the present work, we hypothesized that circ_CLIP2 promoted glioma progression via acting on the miR-641/EPHA3/STAT3 axis.Our research might provide referential molecular markers for clinical diagnosis and treatment for glioma.

Clinical samples collection
In the study, human glioma samples and the corresponding para-cancerous tissues were obtained from 30 glioma patients diagnosed at Guangdong 999 Brain Hospital from 2016 to 2018.The glioma patients in this work were diagnosed pathologically according to the World Health Organization (WHO) certeria.All patients did not receive adjuvant therapy such as chemotherapy or radiotherapy before surgery.The para-cancerous tissues were obtained from the normal tissues (approximately 5 cm away from the cancer tissue) of the same patient.Immediately after removal, all specimens were stored in À80 C until used for experiments.The study was approved by the Ethics Committee of the Guangdong 999 Brain Hospital (No. 2020-010-017).All participants signed a written informed consent form.

Cell transfection
The short hairpin RNA of circ_CLIP2 (sh-circ_CLIP2), the overexpression plasmids of EPHA3 (pcDNA3.1-EPHA3),miR-641 mimics/inhibitor and their negative controls were purchased from GenePharma (Shanghai, China).Glioma cells were seeded in 24-well plates at a density of 3 Â 10 5 cells/well and then incubated at 37 C with 5% CO 2 for 24 h.Then, the above RNAs were transfected into cells by using Lipofectamine TM 3000 (Invitrogen, CA, USA) according to the experimental instructions.After 48h, the transfection efficiency was examined by qRT-PCR.
3-(4, 5-Dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay MTT assay was conducted to assess the changes of LN-229 and U87 cells.In short, cells in each group were obtained and seeded in a 96-well plate (5 Â 10 3 cells/well) for 12 h.At each time point, cells were added 5 mg/mL MTT (Beyotime, Shanghai, China) for 4 h.Then, 100 lL/well DMSO were added and shaken for 15 min.Then the absorbance at 490 nm was analyzed with a microplate reader (Bioteke, Beijing, China).

Cell apoptosis assay
An Annexin-V-fluorescein isothiocyanate (FITC) kit (Sigma, MO, USA) was purchased to examine the apoptotic rate of LN-229 and U87 cells.In short, cells in each group were obtained and washed with PBS twice.After centrifugation, cells (1 Â 10 5 cells) were incubated with 150 lL Annexinbinding buffer (Beyotime, Shanghai, China) containing 10 lL Annexin V-FITC and 5 lL PI stain in the dark for 10 min.Samples were immediately analyzed by flow cytometry (Becton, Dickinson and Company, NJ, USA).

Transwell migration and invasion assay
A transwell chamber (8 mm, Corning, NY, USA) was used to determine the migration and invasion ability of LN-229 and U87 cells.Before the experiment, LN-229 and U87 cells were starved 12 h in a serum-free medium.Then, 200 lL cells at a density of 1 Â 10 4 resuspended in serum-free DMEM were plated in the top chamber and 600 lL of complete medium was placed in the bottom chamber.After 24 h incubation, cells on the upper chamber were removed and further stained with crystal violet for 10 min.Afterward, cells were observed and photographed under a microscope (Nikon).The invasion assay was the same as the migration assay except the upper chamber was precoated with Matrigel (BD, NJ, USA).

Dual luciferase reporter gene assay
The wild-type (WT) or mutant-type (MUT) fragment of human circ_CLIP2 or EPHA3 with/without the binding sites of miR-641 were amplified and inserted into the pmiRGLO vector (Promega, WI, USA).Site-directed mutagenesis was performed using a site mutation kit (Stratagene, CA, USA).To validated the binding relationship between circ_CLIP2 or EPHA3 and miR-641, the recombinant luciferase vectors were co-transfected with miR-641 mimics/inhibitor or mimics/inhibitor NC into cells.After 48 h, the luciferase activity was examined by a dual-luciferase reporter assay system (Promega).

RNA Immunoprecipitation (RIP)
RIP assay was performed using the Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA). 2 Â 10 7 cells were lysed with RIP lysis buffer and incubated with IgG (Abcam, 1:50, ab172730) and Ago2 (Abcam, 1:50, ab186733) antibodies at 4 C overnight.Subsequently, the supernatant was discarded after magnetic beads were washed with wash buffer five times, and then protease K lysate was added to the magnetic beads for lysis at 55 C for 30 min.RNA was purified from the complex, reverse transcribed to cDNA and examined using qRT-PCR.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted with TRIzol (Thermo Fisher Scientific, WI, MA, USA).A cDNA synthesis kit (Toyobo, Tokyo, Japan) and miRNA first-strand cDNA synthesis kit (Sangon, Shanghai, China) were used for the cDNA synthesis of mRNA and miRNA, respectively.Then, SYBR (Thermo Fisher Scientific) was employed for the qRT-PCR assay.b-actin and U6 were used as the reference genes for mRNA and miRNA, respectively.The data was analyzed with the 2 ÀDDCT method.The primers were listed as follows (5 0 -3 0 ):

Statistical analysis
All data were obtained from three independent experiments.Statistical data was analyzed by SPSS 19.0 (IBM, Armonk, NY) and expressed as means ± standards deviation (SD).The differences among two groups or among multiple groups was analyzed by Student's t-tests or one-way analysis of variance (ANOVA) followed by Tukey post-hoc test.The p values less than 0.05 were considered significant.The linear correlation analysis among circ_CLIP2, miR-641 and EPHA3 was performed by Pearson's correlation coefficient analysis.

Results
Circ_CLIP2 and EPHA3 were highly expressed but miR-641 was lowly expressed in glioma Firstly, a total of 30 cases of tumor tissues or matched adjacent tissues obtained from glioma patients were used to examine the expression patterns of circ_CLIP2, miR-641 and EPHA3.qRT-PCR assay showed that circ_CLIP2 and EPHA3 were markedly upregulated compared with that in normal adjacent tissues, while miR-641 expression was markedly reduced (Figure 1(A-D), p < .01,p < .05,p < .001).In addition, Pearson correlation analysis displayed that the expression levels between circ_CLIP2 or EPHA3 and miR-641 exhibited a negative correlation in glioma patients, and the levels between circ_CLIP2 and EPHA3 showed a positive correlation (Figure 1(E-G), p < .01,p < .05,p < .001).

Circ_CLIP2 inhibited miR-641 expression in glioma by adsorbing miR-641
The expression changes of circ_CLIP2 and miR-641 in glioma cells were examined by the qRT-PCR method.As shown in Figure 2(A,B), circ_CLIP2 was significantly upregulated and miR-641 was downregulated in LN-229 and U87 cells compared to that in NHA cells (p < .01,p < .001).Further investigations showed that shcirc_CLIP2 transfection remarkably repressed the level of circ_CLIP2 but increased the level of miR-641 in LN-229 and U87 cells (Figure 2 Starbase software predicted that there was a potential binding site between circ_CLIP2 and miR-641 (Figure 2(E)).The binding relationship between circ_CLIP2 and miR-641 was further validated by dual luciferase reporter and RIP assays.Compared to the anti-normal IgG group, both circ_CLIP2 and miR-641 were markedly expressed in the RNA complexes pulled down by anti-Ago2 (Figure 2(F), p < .001,p < .01,p < .05).Moreover, miR-641 overexpression/knockdown obviously reduced/enhanced the luciferase activity in WT-circ_CLIP2 vectors transfected cells, while there was no significant impact in cells expressing MUT-circ_CLIP2 vectors (Figure 2(G), p < .01).

Circ_CLIP2 promoted cell proliferation, migration and invasion of glioma cells via inhibiting miR-641
To probe the potential roles of the circ_CLIP2/miR-641 axis in regulating the malignant behaviors in glioma cells, LN-229 and U87 cells were co-transfected with sh-circ_CLIP2 and miR-641 inhibitor.qRT-PCR assay showed that miR-641 expression was significantly increased after sh-circ_ CLIP2 transfection, while this change was remarkably reserved by miR-641 inhibitor (Figure 3(A), p < .001,p < .01).MTT assay demonstrated that circ_CLIP2 knockdown dramatically inhibited the viability in LN-229 and U87 cells, while miR-641 silencing attenuated the inhibitory effect of sh-circ_CLIP2 (Figure 3(B), p < .001,p < .01,p < .05).Subsequently, flow cytometry revealed that circ_CLIP2 knockdown strikingly elevated the apoptotic rate of LN-229 and U87 cells, while this phenomenon was significantly abolished in the presence of miR-641 inhibitor (Figure 3(C,D), p < .001,p < .05).Similarly, transwell assay also showed that circ_CLIP2 inhibitory obviously repressed the capabilities of cell migration and invasion in LN-229 and U87 cells, while these impacts were dramatically diminished by miR-641 inhibition (Figure 3(E-H), p < .01,p < .05).

Discussion
Currently, although treatment strategies such as chemotherapy and radiotherapy improve the prognosis of glioma patients, the overall survival of patients is still very short (Cai et al., 2020).With the emergence of specific biomarkers that affect tumor biological functions, exploring the biomarkers of glioma can provide more information for glioma treatment (Verhaak et al., 2010).In the present study, our research revealed that circ_CLIP2 and EPAH3 were upregulated, while miR-641 was downregulated in glioma.Interestingly, the clinical expressions among circ_CLIP2, mIR-641 and EPAH3 in glioma tissues showed a significant correlation, indicating that these three molecules might have a certain connection in the occurrence and development of glioma.
CircRNA is a new type of non-coding RNA molecule discovered in recent years, which can be stably expressed in vivo due to its unique molecular structure and is a kind of biomarker in the progress of various diseases (Zuo et al., 2021).It has been widely described that circRNAs are closely related to the occurrence and development of various human malignancies including glioma.As proof, Peng et al. (2019) revealed that circCPA4 was significantly upregulated in glioma tissues, and its high level was positively related to the poor outcome of glioma.In addition, circNFIX abated glioma cell proliferation and metastasis by upregulating miR-378e and inhibiting RPN2 expression (Ding et al., 2019).Herein, our results revealed that a novel circ_CLIP2 expression was markedly increased in glioma tissues and cells.Moreover, the knockdown of circ_CLIP2 remarkably inhibited glioma cell proliferation, migration and invasion.Similar roles of circ_CLIP2 were also supported by Xiao et al. work (Xiao et al., 2021).Collectively, these results suggested the oncogenic role of circ_CLIP2 in glioma.
A large amount of evidences have confirmed that miR-641 was a tumor suppressor in multiple human malignant tumors.For instance, Yao et al's work revealed that miR-641 was lowly expressed in cervical cancer cells, which could be related to promoter hypermethylation (Yao et al., 2013).Li et al (2022) found that miR-641 repressed cell proliferation but triggered cell apoptosis of breast cancer cells.Similar reports also found in glioma, miR-641 could suppressed the proliferation, migration and invasion of glioma cells by targeting E2F6 (Liu et al., 2021).In the present work, our data found that miR-641 was lowly expressed in glioma tissues and cell lines, and its expression was inversely and significantly correlated with circ_CLIP2 expression.In addition, dual luciferase reporter gene assay and RIP assays validated the direct binding relationship between circ_CLIP2 and miR-641.Additionally, miR-641 inhibition neutralized the inhibitory effects of circ_CLIP2 silencing on the malignant behavior of glioma cells, which further elucidating that miR-641 was a functional target of circ_CLIP2 in glioma.
It's well-known that mature miRNAs can bind to Ago2 to form RNA-induced silencing complexes (RISCs), thereby silencing or degrading their target mRNAs (Ali Syeda et al., 2020).In the current research, our results revealed that miR-641 could directly target to EPHA3 3 0 -UTR, leading to EPHA3 silencing and inactivation of STAT3.EPHA3, as a receptor of Ephs, functions in the germline progression of mammals and in the maintenance of various adult tissues (Janes et al., 2014).EPHA3 dysregulation was associated with various human malignancies (London & Gallo, 2020).Notably, previous literature reported that EphA3 was highly expressed on the tumor-initiating cell population in glioma, and its knockdown reduced tumorigenic potential (Day et al., 2013).Lv et al's study (2018) demonstrated EPHA3 achieved its cancer-promoting effect in gastric cancer by regulating STAT3.Moreover, constitutive activation of STAT3  contributes to the pathogenesis of glioma by promoting both the proliferation and survival of glioma cells (Rahaman et al., 2002).Herein, our results demonstrated that EPHA3 was markedly increased in glioma, and had a negative and positive correlation with miR-641 and circ_CLIP2, respectively.Moreover, circ_CLIP2 could positively activated EPHA3 expression and STAT3 pathway by sponging miR-641.Rescue assays showed that EPHA3 overexpression abrogated the inhibitory effects of miR-624 overexpression on the malignant behaviors of glioma cell by promoting STAT3 expression.Collectively, our data demonstrated that miR-641 overexpression inhibited glioma progression through suppressing the EPHA3/STAT3 pathway.
Overall, our study demonstrated that circ_CLIP2 facilitated the proliferation, migration and invasion of glioma cells by targeting the miR-641/EPHA3/STAT3 axis.The result provides a better understanding of gene-targeted therapy and the prognosis of glioma.However, the number of experimental cases in this study may be insufficient.It is necessary to clarify the expression characteristics of genes in more clinical samples or further study the effects of genes in clinical trials.Moreover, the role of circ_CLIP2/miR-641/EPHA3/STAT3 axis in glioma progression will be further investigated in vivo in future studies.

Figure 1 .
Figure 1.Circ_CLIP2 and EPHA3 were highly expressed but miR-641 was lowly expressed in glioma.(A-C) qRT-PCR was carried out to examine the expressions of circ_CLIP2, niR-641 and EPHA3 expressions in 30 glioma tissues and the normal adjacent tissues.(D) EPHA3 level in glioma tissues and the 30 normal adjacent tissues was analyzed using western blot.(E-G) The expressive correlation amongcirc_CLIP2, miR-641 and EPHA3 were analyzed by Pearson's correlation coefficient analysis.Data were expressed as mean ± SD.N ¼ 3. Ã p < .05,ÃÃ p < .01,ÃÃÃ p < .001.

Figure 2 .
Figure 2. Circ_CLIP2 inhibited miR-641 expression in glioma by adsorbing miR-641.(A,B) The expressions of circ_CLIP2 and miR-641 in LN-229, U87 and NHA cells were assessed by qRT-PCR.(C) qRT-PCR was employed to detect the expressions of circ_CLIP2 and miR-641 LN-229 and U87 cells following sh-NC or sh-circ_CLIP2 transfection.(D) The expression of miR-641 in LN-229 and U87 cells after miR-641 mimics/inhibitor or mimics/inhibitor NC transfection was determined by qRT-PCR.(E) StarBase was employed to predict the binding site between circ_CLIP2 and miR-641.(F,G) The binding relationship between circ_CLIP2 and miR-641 in LN-229 and U87 cells was verified using RIP and dual luciferase reporter gene assays.Data were expressed as mean ± SD.All our data were obtained from three independent experiments.N ¼ 3. Ã p < .05,ÃÃ p < .01,ÃÃÃ p < .001.

Figure 3 .
Figure 3. Circ_CLIP2 promoted cell proliferation, migration and invasion of glioma cells via inhibiting miR-641.LN-229 and U87 cells were silenced circ_CLIP2 alone or co-silenced with sh-circ_CLIP2 and miR-641.After transfection, the cells were obtained for following experiments.(A) qRT-PCR was conducted to examine miR-641 expression.(B) MTT assay was carried out to analyze cell viability.(C,D) Cell apoptosis was examined by flow cytometry.(E-H) Cell migration (E,F) and invasion (G,H) were analyzed by transwell assays.Data were expressed as mean ± SD.All our data were obtained from three independent experiments.N ¼ 3. Ã p < .05,ÃÃ p < .01,ÃÃÃ p < .001.

Figure 4 .
Figure 4. Circ_CLIP2 was a sponge of miR-641 to competitively upregulate EPHA3.(A) Targetscan was employed to predict the binding site between EPHA3 and miR-641.(B-D) The mRNA and protein level of EphA3 in LN-229, U87 and NHA cells was analyzed by qRT-PCR and western blot assay, respectively.(E-G) miR-641 mimics or inhibitor and their respective NCs were transfected into LN-229 and U87 cells, then, the mRNA and protein level of EPHA3 was examined by qRT-PCR and western blot assay (F-G), respectively.(H) The binding relationship between EPHA3 and miR-641 in LN-229 and U87 cells was verified using dual luciferase reporter gene assay.LN-229 and U87 cells were silenced circ_CLIP2 alone or co-silenced with sh-circ_CLIP2 and miR-641.(I) The mRNA level of EPHA3 was examined by qRT-PCR.(J,K) The protein levels of EPHA3, p-STAT3 and STAT3 were detected by western blot.Data were expressed as mean ± SD.All our data were obtained from three independent experiments.N ¼ 3. Ã p < .05,ÃÃ p < .01,ÃÃÃ p < .001.

Figure 5 .
Figure 5. miR-641 suppressed the malignant behaviors of glioma cells through inhibiting EPHA3.(A-C) The mRNA and protein level of EPHA3 in LN-229 and U87 cells after transfection with pcDNA3.1 or pcDNA3.1-EPHAP3transfection was analyzed by qRT-PCR and western blot assays, respectively.LN-229 and U87 cells were overexpressed miR-641 alone or co-overexpressed with miR-641 and EPHA3.After transfection, cells were obtained the following experiments were conducted.(D) MTT assay was carried out to analyze cell viability.(E,F) Cell apoptosis was examined by flow cytometry.(G-J) Cell migration (G,H) and invasion (I,J) were determined using transwell assays.(K,L) EPHA3, STAT3 and p-STAT3 levels in cells were examined by western blot.Data were expressed as mean ± SD.All our data were obtained from three independent experiments.N ¼ 3. Ã p < .05,ÃÃ p < .01,ÃÃÃ p < .001.