miR-637 Prevents Glioblastoma Progression by Interrupting ZEB2/WNT/β-catenin Cascades

Glioblastomas (GBMs) are the most frequent primary malignancies in the central nervous system. Aberrant activation of WNT/β-catenin signaling pathways is critical for GBM malignancy. However, the regulation of WNT/β-catenin signaling cascades remains unclear. Presently, we observed the increased expression of ZEB2 and the decreased expression of miR-637 in GBM. The expression of miR-637 was negatively correlated with ZEB2 expression. miR-637 overexpression overcame the ZEB2-enhanced cell proliferation and G1/S phase transition. Besides, miR-637 suppressed the canonical WNT/β-catenin pathways by targeting WNT7A directly. Gain- and loss-of-function experiments with U251 mice demonstrated that miR-637 inhibited cell proliferation and arrested the G1/S phase transition, leading to tumor growth suppression. The collective findings suggest that ZEB2 and WNT/β-catenin cascades merge at miR-637, and the ectopic expression of miR-637 disturbs ZEB2/WNT/β-catenin-mediated GBM growth. The findings provide new clues for improving β-catenin-targeted therapy against GBM.


Introduction
Gliomas are the most frequent primary malignancies in the central nervous system. According to their histopathological characteristics, gliomas are categorized as grades I, II, III, and IV (glioblastoma, GBM) according to the World Health Organization (WHO) classification for central nervous system tumors (Chen et al. 2017). Histologic criteria are essential for the diagnosis of gliomas. Molecular characteristics have been introduced into the classification system to refine the diagnostic standard, prognostic markers, and the initial options for targeted therapies (Reifenberger et al. 2017). These characteristics include MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation, IDH (isocitrate dehydrogenase) mutations, chromosome 1p/19q deletion, and EGFR (epidermal growth factor receptor) amplification. Particularly, mounting evidence has demonstrated that WNT/β-catenin signaling pathways are frequently hyperactive in GBM and are promising therapeutic targets against GBM progression (He et al. 2019). However, the dysregulation of WNT/β-catenin in GBM has not been thoroughly investigated.
Zinc finger E-box binding homeobox 2 (ZEB2) associates with the 5'-CACCT -3' sequence in various promoters and inhibits transcription. ZEB2 has been implicated in multiple biological processes, including development, differentiation, and tumor progression. ZEB2 represses E-cadherin and causes epithelial-mesenchymal transition along with Snail and Slug (Nagaraja and Nagarajan 2018). Besides, ZEB2 activates WNT/β-catenin signaling pathways and contributes to cancer progression . Accumulating evidence suggests that the dysregulation of ZEB2 is involved in angiogenesis, proliferation, invasion, and metastasis of GBM. For instance, Chen et al. demonstrated that ZEB2 is an independent prognostic factor and that its overexpression is correlated with an unfavorable prognosis of GBM . Feng et al. reported that TRIM14 promoted the motility of GBM cells in a ZEB2-dependent manner (Feng et al. 2019). Furthermore, the MSH6-CXCR4-TGFB1 1 3 complex promotes ZEB2 transcription, leading to oncogenesis and tumor progression (Chen et al. 2019). miR-590 targets ZEB2 and ZEB1, inhibiting the invasiveness of GBM (Pang et al. 2015). Despite these observations, the details of ZEB2-mediated signaling cascades in primary GBM are not fully understood.
MicroRNAs (miRNAs) are noncoding RNAs that contain less than 25 nucleotides. miRNAs play pivotal roles in different physical and pathological processes, including cell growth, apoptosis, and survival. Abnormal regulation of miRNAs is involved in developing human cancer, including GBM (Saadatpour et al. 2016). Different miRNA expression profiles were used to classify the GBM sub-groups. For instance, Zeng and colleagues demonstrated that miR-17-5p and CXCL14 have potential predictive values for higher-grade gliomas (Zeng et al. 2018). Furthermore, an increasing number of miRNAs were identified as independent prognostic factors. A low miR-504:FZD7 expression ratio has been correlated with poor clinical outcomes against chemotherapy and radiotherapy for mesenchymal subtype GBM ). Moreover, miRNAs are able to predict response to therapy. Huang et al. described the ability of miR-93 to inhibit autophagy and suggested the involvement of miR-93 in the sensitivity to the therapeutic combination of irradiation and temozolomide (Huang et al. 2019).  in a long noncoding RNA (lncRNA)-miRNA cluster that predicted the survival time of patients with wild-type BRCA1/2-subtype ovarian cancer (Guo et al. 2017). miR-637 forms a complex with various noncoding RNAs and mRNAs, and mediates oncogenesis, proliferation, chemoresistance, and metastasis. For instance, Zhang et al. demonstrated that the interplays of the lncRNA LINC00473:miR-637:CDK6 regulated glioma proliferation and invasion (Zhang et al. 2019b). Que et al. revealed that the decreased miR-637 expression was related to shorter survival of patients with gliomas and described that overexpression of miR-637 induced tumor regression (Que et al. 2015). However, the mechanisms underlying miR-637-mediated rehabilitation remain unclear. Further investigation of miR-637 in the regulation of progression and relapse of GBM may grant novel druggable targets.
In the present study, we aimed to illustrate the expression of miR-637 in ZEB2-deficient GBM and the correlations between miR-637 and ZEB2. We attempted to uncover the mechanisms underlying the ZEB2:miR-637:WNT axis-regulated GBM malignancies. The present results may provide novel insights into treatments for advanced GBM.

Patients and Sample Collection
The study was approved by the Ethics Committee of the First Hospital of China Medical University. Seventy-one patients with different grades of gliomas were recruited. All participants provided written informed consent for research purposes and publication. Independent pathologists reviewed specimen slides to validate the histological subtypes and WHO grade of gliomas (Table 1). The specimens were kept in liquid nitrogen.

Analysis of miRNA and mRNA Associations
The interactions between miRNA and mRNA were predicted by the miRDB database (Agarwal et al. 2015; Wang 2020) following the site instructions.

Cell Proliferation Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure cell proliferation. Cells were seeded (1 × 10 4 /well) in 96-well plates with 0.1 ml of medium and allowed to attach overnight. Cells were transfected with plasmids or RNA oligonucleotides according to the manufacturer' s protocols (Sigma-Aldrich, St. Louis, MO, USA). Forty-eight hours later, the cells were exposed to medium supplemented with 10 μl MTT solution (Sigma-Aldrich, #M2128; 5 mg/ml) for 4 h at 37 °C. The medium was replaced by 150 μl of a solution of dimethyl sulfoxide (Sigma-Aldrich; #472,301) solution, followed by incubation for 10 min. The absorbance at 490 nm was analyzed using a microplate reader (Life Science, Hercules, CA, USA).

Flow Cytometry Analysis
U251 cells (6 × 10 5 ) were plated in six-well petri dishes prior to analysis. Twenty-four hours later, cells were collected and fixed in 70% pre-chilled ethanol (-20 °C) and incubated at 4 °C overnight. Cells were gently resuspended in PBS with 50 µg/mL ribonuclease (#R6513), followed by incubation at 37 °C for one h. Afterward, cells were treated with propidium iodide solution (#P4864, Sigma-Aldrich) at a final concentration of 10 µg/ml for 5 min. Cell cycle distribution was detected by flow cytometry using a FACS-Calibur™ device following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Analysis of flow cytometry data was performed using CellQuest Pro software (version 5.1; BD Biosciences). Differences among groups were estimated by two-way ANOVA, followed by a Tukey's multiple comparisons test. P < 0.05 was considered statistically significant. Original flow cytometry analysis data were available if required.

Luciferase Reporter Assay
Cells (4 × 10 4 ) per well of a 24-well plate were transfected with wild-type or mutant WNT7A 3'-UTR together with miR-637 or the vector using TurboFectin Transfection Reagent (#TF81001, OriGene). Twenty-four hours later, the luciferase signal was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's protocol. Data were collected using the iMark™ Microplate Absorbance Reader 1 3 (Bio-Rad) and were analyzed by two-way ANOVA followed by Sidak's multiple comparisons test. P < 0.05 was considered statistically significant. The relative luciferase activity is presented as values of each group normalized to the group WNT7A plus vector values.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using the miRNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Five hundred nanograms RNA was reverse transcribed to cDNA using a QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed using the Mx3000P real-time PCR system (Thermo Fisher Scientific). PCR was performed using 40 cycles of 94 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s. All experiments were independently performed three times. Gene expression was normalized to GAPDH to analyze the relative expression using the previously described 2 −ΔΔCq method (Livak and Schmittgen 2001). The primers for detecting genes were generated by Santa Cruz Biotechnology. The

U251 Mouse Model
The animal study was approved by the Institutional Animal Care and Use Committee of the First Hospital of China Medical University. Twenty-four 4-week-old female nude. BALB/c mice (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were maintained under specific-pathogen-free conditions. U251 cells (5 × 10 6 ) were subcutaneously injected into the flanks of the mice. Seven days later, mice with tumors were randomized into 3 groups (n = 6 per group) when the mean tumor volume approached 100 mm 3 . Tumor volumes were calculated as V = 0.5 × L × W 2 , where V is tumor volume, L is tumor length, and W is tumor width. Tumor volumes were measured every four days. The mice were euthanized on day 28. Tumors were removed, weighed, and recorded using standard protocols.

Statistical Analyses
The data from all experiments are presented as means ± standard deviations. The association of gene expression was analyzed using Spearman's correlation test. Group differences were evaluated by ordinary one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test. P < 0.05 is statistically significant. Statistical analysis was performed using GraphPad version 8.0 (San Diego, CA, USA).

ZEB2 Increases in GBM and Contributes to Cell Proliferation and G1/S Phase Progression
ZEB2 expression in 18 grade II, 13 grade III, and 40 grade IV (GBM) specimens were examined. ZEB2 increased in GBM compared to adjacent tissues, while that of grade II and III was similar to adjacent tissues (Fig. 1a). There were no significant differences in ZEB2 expression among various histological subtypes of gliomas as well as gender and age (Table 1). Moreover, ZEB2 expression was higher at the translation and transcriptional levels in GBM cell lines than in NHA (Fig. 1b). The findings agreed with the ZEB2 alterations in tissues. To further investigate the effect of ZEB2 on cell proliferation, we generated stable ZEB2-knockdown U251 and LN229 sublines (Fig. 1c). The downregulation of ZEB2 impaired cell proliferation significantly compared to the sh-NC (Fig. 1d). We selected the sh-ZEB#2 subline to analyze the cell cycle distribution because there was an insignificant difference in the growth curves of the sublines. As expected, flow cytometry analysis revealed that ZEB2-knockdown caused G1/S phase arrest (Fig. 1e).

miR-637 Suppresses Cell Proliferation and Induces G1/S Arrest in the Absence of ZEB2
We conducted miRNA arrays with the sublines and control cells to investigate the alteration of the miRNA profile of ZEB2-knockdown GBM cells. The heatmaps in Fig. 2a displayed the distinctly expressed miRNA, where miR-637 showed the most considerable reduction. We also determined the expression of several miRNA candidates in sh-ZEB2 knockdown sublines (data not shown). The results were consistent with those shown in Fig. 2A. We, therefore, explored miR-637 expression in GBM cells and tissues. The expression of miR-637 declined in GBM cells compared to that in NHA (Fig. 2b). Moreover, miR-637 expression was significantly lower in GBM specimens than in adjacent tissues, while that of grade II and III was consistent with adjacent tissues (Fig. 2c). The differences in miR-637 expression among histological subtypes were insignificant in chi-square analysis (Table 1). A negative correlation between miR-637 and ZEB2 expression was revealed by Spearman's test (Fig. 2d). The R 2 value of 0.5605 indicated that the relationship between miR637 and ZEB2 accounted for half of the changes in miR-637 expression. The downregulation of ZEB2 promoted miR-637 (Fig. 2e), which agreed with the results in Fig. 2d.
To determine the effects of miR-637 on cell proliferation, we introduced miR-637 mimics (miR-637), miR-637 anti-miRNA oligonucleotides (miR-637AMO), and a combination of miR-637AMO and sh-ZEB2#1 into GBM cells. The results in Fig. 2f suggested that miR-637 expression increased in cells expressing miR-637 mimics and decreased significantly in cells expressing miR-637AMO. ZEB2 decreased markedly in cells expressing the combination. The overexpression of miR-637 suppressed cell proliferation, while the downregulation of miR-637 strikingly induced proliferation. Notably, the proliferation of ZEB2/miR637-dual knockdown cells returned to the control level (Fig. 2g). Flow cytometry analysis showed that the percentage of the G1 phase in cells expressing miR-637 was approximately 70%, while that in cells lacking miR-637 was < 40% compared to that in control cells (slightly > 50%). The results suggested that the overexpression of miR-637 attenuated the G1/S phase transition, while miR-637 knockdown enhanced G1/S phase progression. The percentage of the G1 phase in cells lacking miR-637 and ZEB2 showed little change compared to the control (Fig. 2h).
The depletion of ZEB2 enhanced miR-637 expression in GBM cells and the ZEB2-miR637 axis was involved in regulating cell proliferation and G1/S transition.

miR-637 Inhibits Cell Proliferation and G1/S Phase Transition by Targeting WNT7A
As miRNA participated in regulating tumor progression by targeting various mRNA candidates, we employed the miRDB database to predict the miRNA-mRNA interactions. Among a few candidate targets of miR-637, WNT7A expression showed the most significant augmentation in cells that lacked miR-637 (Supplementary Figure d). Moreover, TNMplot database showed WNT7A expression increased in GBM compared to non-cancerous brain samples (Bartha and Gyorffy 2021), where RNA sequencing data were analyzed using Mann-Whitney tests. Thus, WNT7A was selected for further investigation.
WNT7A was elevated in GBM cells compared with NHA (Fig. 3a). Moreover, WNT7A expression was increased in GBM compared to adjacent tissues, whereas WNT7A expression in grade II and III remained consistent with that in adjacent tissues (Fig. 3b). The WNT7A expression among various histological subtypes remained stable (Table 1). WNT7A expression was negatively correlated with miR-637 by Spearman's rank correlation analysis (Fig. 3c). The R 2 value of 0.5304 suggested that approximately half of the WNT7A alteration was triggered by miR-637. We Fig. 1 ZEB2 increases in GBM and contributes to cell proliferation and G1/S phase progression. A The expression of ZEB2 in adjacent and gliomas tissues. Adj, adjacent tissues; Grade II, III, IV, WHO grade of gliomas. *P < 0.05, vs. Adj; ns, no significance, vs. Adj. B ZEB2 expression in normal human astrocytes (NHA) and GBM cell lines was detected by western blot and qRT-PCR, respectively. *P < 0.05, vs. NHA. C The depletion of ZEB2 in GBM cells was validated by western blot. D The proliferation of the indicated cells was determined by MTT assay. *P < 0.05, vs. sh-NC. E The cell cycle distribution of the indicated cells was detected by flow cytometry. *P < 0.05, vs. sh-NC. Results represent the mean ± SD of three independent experiments. sh-NC, short hairpin RNA scrambled negative control generated the WNT7A 3'UTR mutation (WNT7A MUT) in which the scrambled nucleotides were insufficient for the binding of miR-637. The luciferase activity of WNT7A 3'UTR dropped significantly, while that of WNT7A MUT 3'UTR only changed marginally (Fig. 3d). The ectopic expression of miR-637AMO repressed miR-637 expression (Fig. 3e) and promoted the expression of WNT7A (Fig. 3f). Because WNT7A was one of the target genes of miR-637, we wondered whether miR-637 repressed cell proliferation and retarded G1/S phase transition in a WNT7A-dependent manner. Further exploration showed that the silencing of WNT7A overcame miR-637AMO-promoted cell proliferation (Fig. 3g). In addition, the silencing of WNT7A reversed the miR-637AMO-enhanced G1/S phase progression. The results in Fig. 3h indicated that the population of the G1 phase in cells lacked miR-637 and WNT7A returned the proportion to that of the control. The collective findings indicated that miR-637 suppressed cell proliferation and hampered G1/S transition by targeting WNT7A.

miR-637 Inhibits Cell Proliferation and G1/S Phase Transition by Interrupting WNT/β-Catenin Signaling Pathways
The WNT family is essential in activating the canonical WNT/β-catenin signaling cascades, including WNT7A (Noda et al. 2016). We introduced β-catenin and shRNA targeting β-catenin into cells in the absence of miR-637. The downregulation of miR-637 enhanced WNT7A and β-catenin expression, while sh-β-catenin significantly inhibited β-catenin expression (Fig. 4a). Further investigation demonstrated that sh-β-catenin abolished the miR-637AMOboosted cell proliferation (Fig. 4b) and β-catenin abrogated miR-637AMO-induced G1/S progression (Fig. 4c). Previous studies have established that active β-catenin binds to T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family members and triggers the transcription of target gene CCND1, which encodes the critical regulator of G1/S phase transition (Shang et al. 2017). Thus, the luciferase activity of TCF/LEF binding sites is an indicator of β-catenin-mediated transcriptional activation. We, therefore, introduced Tcf/ Lef-luciferase or CCND1-luciferase with miR-637AMO, or a combination of miR-637AMO and sh-β-catenin into cells. As shown in Fig. 4d, miR-637AMO significantly enhanced the luciferase activity of TCF/LEF compared to the control group, whereas the ectopic expression of sh-βcatenin defeated the miR-637AMO-mediated promotion. The changes in CCND1-luciferase were similar to those of TCF/LEF luciferase (Fig. 4e). The results in Fig. 4 suggested that miR-637 blocked cell proliferation and cell cycle progression via WNT/β-catenin signaling pathways.

miR-637 Impedes Tumor Growth in U251 Mouse Models
To illustrate the effects of miR-637 on tumor growth, we established human U251 animal models by subcutaneous injection of U251 cells. Tumor volumes of the miR-637 group declined, whereas those of miR-637AMO increased (Fig. 5a). Notably, the tumor volumes of the dual-knockdown group were almost the same as those of the control group. The findings suggested that β-catenin knockdown abrogated miR-637AMO-enhanced tumor growth. Moreover, tumor weights in the miR-637 group decreased, while those in miR-637AMO increased compared to the control group. The differences in tumor weights between the dual knockdown and control groups were insignificant, consistent with the changes in growth curves (Fig. 5b). We further examined the expression of β-catenin and caspase 3 to explore apoptosis. β-catenin elevated in the miR-637AMO group. Inversely, β-catenin was reduced in the miR-637 and sh-β-catenin groups compared to the control (Fig. 5c). miR-637 and the dual blockage of miR-637 and β-catenin promoted caspase 3, whereas miR-637AMO repressed the expression of caspase 3 compared to the control group. The expression of WNT7A and β-catenin decreased in the miR-637 group while increasing in the miR-637AMO group compared to the control group ( Fig. 5d and e). WNT7A expression remained stable in the β-catenin-knockdown group compared to the miR-637AMO, indicating that miR-637 targeted WNT7A and hampered WNT/β-catenin signaling pathways. We also determined Cyclin D1, which can be promoted by β-catenin and subsequently promotes the G1/S phase transition (Shang et al. 2017). miR-637AMO enhanced Cyclin D1 expression, while miR-637 and the dual knockdown attenuated Cyclin D1 compared to the control group, consistent with the alteration of β-catenin (Fig. 5e). The collective results in Fig. 5 proved that miR-637 retarded tumor growth by restricting the WNT/β-catenin cascades.

Discussion
ZEB2 is characterized by two-handed zinc fingers, which associate with the 5'-CACCT-3' sequence within different promoters and repress transcription, including E-cadherin. Chen et al. demonstrated that ZEB2 overexpression resulted in a shorter overall survival of GBM patients ). Suzuki et al. found that ZEB2 expression increased significantly in grade IV gliomas (GBM), whereas ZEB2 expression was slightly higher in grade II gliomas. ZEB2 is positively correlated with GBM invasiveness (Suzuki et al. 2018). The present results that GBM features ZEB2 1 3 overexpression, while grade II and grade III showed similar ZEB2 expression compared to non-cancerous tissues, are consistent with these previous studies.
ZEB2 cooperates with lncRNA, miRNA, and protein. For instance, ZEB2 inhibited E-cadherin, while enhancing N-cadherin and vimentin, leading to epithelial to mesenchymal transition (Vu and Datta 2017). The inhibition of ZEB2 transcription promoted GBM progression (Myung et al. 2021). Chen et al. showed an oncogenic loop of MSH6:CXCR4:TGFB1 conferred GBM resistance against temozolomide in a ZEB2-dependent manner (Chen et al. 2019). Cheng et al. validated that lncRNA MALAT1 released ZEB2 from miR-124, accelerating GBM growth (Cheng et al. 2020). The details of the interplay between ZEB2 and WNT/β-catenin remain controversial. Comijn et al. demonstrated that either the location or the expression of β-catenin was altered after SIP1 (ZEB2) induction in colorectal cancer cells (Comijn et al. 2001). Qi et al. argued that the knockdown of ZEB2 suppressed β-catenin expression in glioma cells (Qi et al. 2012). Zhang et al. illustrated that miR-498 targeted ZEB2 and XIAP, hindered liver cancer malignant behavior via inactivation of WNT/β-catenin pathways (Zhang et al. 2019c). Zhang and colleagues proved that the ZEB2/miR-200c loop participated in prostate carcinoma progression (Zhang et al. 2019a). We found miR-637 was a new interchange of ZEB2 and the canonical WNT/βcatenin pathways, which are frequently activated in GBM progression. The finding provides an attractive therapeutic target against GBM.
In the current study, we found that miR-637 expression declined in GBM, which was in agreement with previous research Zhang et al. 2019b). Moreover, the present results that miR-637 presented in grade II and grade III gliomas marginally suggested that miR-637 showed potential as a predictive index for better GBM prognosis. Large samples covering different grade subtypes or diverse genetic characteristics of GBM could be helpful in the future.
In addition, we found ZEB2 and WNT/β-catenin pathways merged in miR-637. The ectopic expression of miR-637 interrupted the collaboration of ZEB2 and β-catenin, leading to the inhibition of GBM growth. Previous studies have shown methods for investigating ZEB2 and miRNAs at the transcriptional level. Cameron et al. conducted endogenous chromatin immunoprecipitation assays to identify the binding regions of ZEB1/2 and miR-200b promoter according to the prediction (Bracken et al. 2008). Ren et al. demonstrated that ZEB2 attached to the first E-box in the miR-145 promoter using the same methods (Ren et al. 2014). Likely, the promoter sequences of miR-637 can be found by recruiting ENSEMBL (http:// www. ensem bl. org/ index. html). Very recently, Zeng et al. identified the binding regions between ZEB2 and miR-637 promoter by performing chromatin immunoprecipitation coupled with dual-luciferase reporter assay (Zeng et al. 2021).
Previous studies have revealed that miR-637 regulated gliomas progression by targeting multiple genes. Qiu et al. revealed that miR-637 targeted Akt1 3'UTR. The reduction of miR-637 indicated a favorable outcome of gliomas (Que et al. 2015).  axis was interrupted by LINC00473, leading to the inhibition of proliferation and invasion of glioma cells (Zhang et al. 2019b). Jin et al. proved that the interplays of cir-cRNA EPHB4:miR-637:SOX10 participated in maintaining the stem properties of gliomas (Jin et al. 2021). Recently, Zeng et al. found ZEB2 suppressed miR-637 and rescued HMGA1, promoting gliomas malignancy in a vimentindependent manner (Zeng et al. 2021). The present findings extend the knowledge of miR-637 regulating gliomas progression. Further investigation with different stages or subtypes of gliomas samples, including primary gliomas and recurrent gliomas, is important for clarifying the discrepancy of miR-637 targets. Besides, conducting experiments with cells that possessed specific mutants, such as IDH1 and EGFR, may help determine the dominant signaling pathways in gliomas progression.
Mounting evidence suggested that the crosstalk between PI3K/AKT pathways and WNT/β-catenin pathways was pivotal for maintaining the malignancy of gliomas, although the mechanisms were not fully understood. For instance, the inhibition of AKT by small molecular inhibitors and noncoding RNAs suppressed glioma progression via partially repressed WNT/β-catenin pathways (Vallee et al. 2018;Han et al. 2010). Zhang et al. demonstrated that N-cadherin activated β-catenin by phosphorylated AKT1 and GSK3β cooperatively (Zhang et al. 2013), contributing to neuronal differentiation and cell survival. Moreover, Qiu et al. revealed that miR-637 targeted AKT1 3'UTR, indicating that the overexpression of miR-637 was a promising therapeutic intervention against gliomas (Que et al. 2015). Li et al. demonstrated that miR-637 targeted AKT1 directly and Fig. 2 miR-637 increases in ZEB2 knockdown GBM, leading to reduced cell proliferation and arrest of G1/S transition. A The differential expression of miRNAs in the indicated cells was analyzed by miRNA microarray. The color indicates the fold-change of target genes compared to housekeeping genes in each group. B miR-637 expression in GBM cell lines was examined by qRT-PCR. *P < 0.05, vs. NHA. C miR-637 expression in adjacent or glioma tissues was examined by qRT-PCR. *P < 0.05, vs. Adj; ns, no significance, vs. Adj. D The correlation of ZEB2 and miR-637 expression was estimated by the Spearman correlation test. P < 0.05 indicated statistical significance. E The expression of miR-637 in the indicated cells was analyzed by qRT-PCR. *P < 0.05, vs. sh-NC. F The expression of miR-637 and ZEB2 in the indicated cells was detected by qRT-PCR. *P < 0.05, vs. Control. ns, no significance. G The proliferation of the indicated cells was determined by MTT assay. *P < 0.05, vs.
Control. H The cell cycle distribution of the indicated cells was analyzed by flow cytometry. *P < 0.05, vs. Control. Results represent the mean ± SD of three independent experiments ◂ 1 3 Fig. 3 miR-637 inhibits cell proliferation and cell cycle progression by targeting WNT7A. A WNT7A expression in glioma cell lines was examined by western blot. B WNT7A expression in adjacent or gliomas tissues was examined by qRT-PCR. Adj, adjacent tissues; Grade II, III, IV, WHO grade of gliomas. *P < 0.05, vs. Adj; ns, no significance, vs. Adj. C The correlation of WNT7A and miR-637 was estimated by Spearman correlation analysis. P < 0.05 indicated statistical significance. D Schematic of the binding sites between ZEB2 and miR-637 and the relative luciferase activity of WNT7A determined by dual-luciferase assay. *P < 0.05, vs. vector. E The expression of miR-637 and WNT7A in cells analyzed by qRT-PCR. *P < 0.05, vs. Control. F Expression of WNT7A in cells analyzed by western blot. G The proliferation of the indicated cells was determined by the MTT assay. *P < 0.05, vs. Control. H The cell cycle distribution of the indicated cells was analyzed by flow cytometry. *P < 0.05, vs. Control. Results represent the mean ± SD of three independent experiments Fig. 4 miR-637 suppresses proliferation and induces G1/S phase arrest by interrupting WNT/β-catenin signaling. A The indicated genes expression in the indicated cells was determined by qRT-PCR. *P < 0.05, vs. Control. *P < 0.05, vs. Control. B The proliferation of the indicated cells was determined by the MTT assay. *P < 0.05, vs. Control. C The cell cycle distribution of the indicated cells was analyzed by flow cytometry. *P < 0.05, vs. Control; ns, vs. Control. ns, no significance. D, E The relative luciferase activity of TCF/LEF or CCND1 3'UTR was assessed by the dual-luciferase reporter system. * P < 0.05, vs. Control. # P < 0.05, vs. miR-637AMO. sh-β-Cat, shRNA targeting β-catenin The tumor growth curves were recorded during treatment. *P < 0.05, vs. Control; ns, vs. Control. ns, no significance. B The tumor weights were measured post-treatment. *P < 0.05, vs. Control; ns, vs. Control. ns, no significance. C The expression of β-catenin and Caspase hampered cell cycle progression . We found the binding sequences of miR-637 and WNT7A were identical with those of miR-637 and AKT1. The similarity implied that miR-637 targeted AKT1 and WNT7A competitively. The possibility that miR-637 suppressed AKT and subsequently inhibited β-catenin cannot be excluded. To date, few β-catenin-specific inhibitors have been evaluated in GBMrelated clinical trials, although many compounds have been shown to antagonize aberrant β-catenin activity. The reasons for the discrepancy in the crosstalk between ZEB2 and β-catenin across various cancers remain unknown. Further experiments to clarify the mechanisms underlying ZEB2 repressed miR-637 are necessary.
WNT7A is required to maintain the normal functions of blood-brain barrier (Reis et al. 2012;Chang et al. 2017). Although the interplays of ZEB2:miR-637:WNT7A regulated glioma progression, the role of the interactions in endothelial cells is unknown. Uncovering the mechanisms of miR-637/WNT7A in endothelial cells, as well as the interactions of glioma cells and endothelial cells, may provide a rationale for developing new therapeutic interventions against GBM. Further exploration with miR-637 knockdown or hyperactivating ZEB2 endothelial cells is necessary for filling the gap.
Briefly, we found that the downregulation of ZEB2 can promote miR-637 expression in GBM. miR-637 overexpression suppresses the hyperactivation of WNT/β-catenin signaling cascades, hampering GBM proliferation. The ectopic expression of miR-637 can block the crosstalk between ZEB2 and WNT/β-catenin signaling pathways. The findings provide new insights into the mechanisms underlying ZEB2 targeting the WNT/β-catenin pathways. The findings could inform improved β-catenin-targeted therapy for GBM.