LncRNA SPRY4‐IT1 facilitates cell proliferation and angiogenesis of glioma via the miR‐101‐3p/EZH2/VEGFA signaling axis

Abstract 
Background
 SPRY4‐IT1 (SPRY4 intronic transcript 1) is a long non‐coding RNA (lncRNA) that has been identified as a novel oncogene in various cancers, including glioma. However, its function and underlying mechanism in glioma remain largely unclear. Here, we investigated the role of SPRY4‐IT1 in the development of glioma and its underlying mechanism. Methods Bioinformatics analysis and RT‐qPCR assay were used to examine the expression of SPRY4‐IT1 in glioma tissues. The CCK‐8, EdU, and Xenograft tumor assays wereperformed to assess the proliferation effect of glioma cells. The tube forming assay and Chick Embryo Chorioallantoic Membrane (CAM) assay were conducted to detect the angiogenesis effect of HUVECs. RNA‐sequencing, western blotting, RT‐qPCR, ELISA, and IHC assays were employed to verify the regulatory mechanism of the SPRY4‐IT1/ miR‐101‐3p/EZH2/VEGFA axis. Results Analysis of the TCGA dataset and data from our own cohort demonstrated that SPRY4‐IT1 was overexpressed in patients with glioma, and high SPRY4‐IT1 expression correlated with poor prognosis. In vitro and in vivo experiments showed that SPRY4‐IT1 promoted the proliferation of glioma cells. RNA sequencing and Gene Ontology (GO) enrichment analysis indicated significant enrichment of angiogenesis. HUVEC tube forming assay and CAM assay confirmed that SPRY4‐IT1 could induce angiogenesis of glioma cells in vitro and in vivo. Mechanistically, SPRY4‐IT1 upregulated EZH2 expression by sponging miR‐101‐3p to induce VEGFA expression in glioma cells. Moreover, SPRY4‐IT1 activated the VEGFR2/AKT/ERK1/2 pathway in HUVECs mediated by glioma cells. Rescue experiments further confirmed that SPRY4‐IT1 promoted glioma cell proliferation and angiogenesis via the miR‐101‐3p/EZH2/VEGFA signaling axis. Conclusions Our findings provide compelling evidence showing that SPRY4‐IT1 upregulated EZH2 to induce VEGFA by sponging miR‐101‐3p, thereby achieving cell proliferation and angiogenesis in glioma. Therefore, targeting SPRY4‐IT1/miR‐101‐3p/EZH2/VEGFA axis may improve the outcomes of patients with glioma.


| INTRODUCTION
Glioma, the most common primary malignant tumor of the central nervous system (CNS), represents nearly 25.13% of all primary CNS tumors. Glioblastoma (GBM) is the most aggressive type and most common form of glioma in adults, accounting for 57.7% of all gliomas. 1 Despite current advances in multimodal therapy (surgery, radiotherapy, and chemotherapy) for GBM, GBM patients still suffer from poor prognoses (5-year survival rate of only 5.8%). 2 Therefore, alternative approaches to improve the treatment of glioma are urgently needed.
The vast majority of the human genome is transcribed. However, approximately 98% of transcripts do not encode for any proteins and are termed non-coding RNAs (ncRNAs). Long non-coding RNA (lncRNA) accounts for approximately 80%-90% of the ncRNAs, has transcripts longer than 200 nucleotides and was once regarded as junk DNA. 3 However, lncRNA has been increasingly recognized to play large regulatory roles in the biological processes. 4 To date, dysregulation of lncRNA has been linked to every cancer investigated and affects all major cancer hallmarks. 5,6 Additionally, increasing evidence has indicated that lncRNA plays a critical role in the tumorigenesis, progression, and drug resistance of glioma. 7 SPRY4-IT1 (SPRY4 intronic transcript 1), derived from an intron region within the SPRY4 gene, which was confirmed to be localized in the cytoplasm of tumor cells, has attracted increasing attention among various lncRNA. [8][9][10] SPRY4-IT1 was first discovered as an oncogene modulating melanoma cell apoptosis and invasion. 8 In recent years, genome-wide analysis and molecular functional studies have focused on the role of SPRY4-IT1 in cancer progression. 9,11,12 Shojaei et al. found that SPRY4-IT1 might be a novel oncogenic lncRNA by reviewing the biological function and mechanism of SPRY4-IT1 in cancer progression. 13 Increasing evidence has also suggested SPRY4-IT1 as a prognostic factor for poor outcomes as it was associated with accelerating glioma cell proliferation, invasion, and migration via the epithelial-mesenchymal transition. 14,15 However, the mechanism of SPRY4-IT1 in glioma remains unclear.
In this current study, we assessed the prognostic value of SPRY4-IT1 in patients with glioma and explored its underlying molecular mechanisms in glioma cells and animal models. We found that SPRY4-IT1 was upregulated in glioma specimens and correlated with a poor prognosis in glioma patients. Moreover, SPRY4-IT1 could promote glioma cell proliferation and glioma cell-induced angiogenesis in vitro and in vivo. Further, we confirmed that SPRY4-IT1 regulated the miR-101-3p/EZH2/VEGFA signaling axis to induce tumorigenesis and angiogenesis in glioma. Taken together, our findings provided insights into the role, prognostic significance and underlying mechanism of SPRY4-IT1 in glioma.

| Clinical samples
Fifty-nine samples of human glioma tissues (WHO II grade: 15 samples; WHO III grade: 21 samples; WHO IV grade: 23 samples) were used in this study. They were both pathologically diagnosed as gliomas for the first time from February 2017 to September 2020 at the Department of Neurosurgery, the Second Affiliated Hospital of Soochow University (Suzhou, Jiangsu, China). They did not receive any chemotherapy or radiotherapy before surgery. Their clinicopathological features are shown in Table S1. Another dataset containing three samples from brain trauma patients was collected as the control group. All patients/participants provided written informed consent to participate in this study.

| Cell culture
Human glioma cell lines (U87, SNB19, and U118) and normal human astrocytes (NHA) were provided by Dr.

| Lentivirus packaging and generation of stable cells
The short hairpin RNA of SPRY4-IT1 (shSPRY4-IT1) (CCGGGGTGGTTGAAAGGAATCCTCT CGAGAGG ATTCCTTTCAACCACCTTTTTG) was inserted into the hU6-MCS-Ubiquitin-IRES-puro plasmid. Lentivirus packaging was performed according to a previously described method. 16 HEK 293T cells were co-transfected with a shSPRY4-IT1 plasmid, Pcmvdr8.91 plasmid, and pMD.G-VSV-G plasmid. The cell supernatants were harvested and filtered with a 0.45 μm nitrocellulose filter 48 h post-transfection. Finally, the glioma cells were infected with the supernatants for 48 h and screened with 5 μg/mL puromycin for 1-2 weeks to obtain stable cells.

| Collection of the glioma conditioned medium (GCM)
The collection of GCM was performed as previously mentioned. 17 The glioma cells were grown to 80% confluence under different treatments, and the culture medium was changed with fresh serum-free DMEM for 24 h. Then, the supernatants were harvested, centrifuged at 2000 g for 10 min at 4°C to remove cellular debris, and supplemented with 0.5% fresh serum. The GCM was immediately used for subsequent experiments.

| EdU assay
Cell proliferation was assessed using an EdU kit (RiboBio, Guangzhou, China) following the manufacturer's protocol. The glioma cells were incubated with EdU solution for 4 h, then dyed with Apollo Dye solution after being fixed with 4% paraformaldehyde (PFA). Photographs of EdU cells were captured with a confocal microscope (Zeiss), and the EdU-positive cell rate was quantified using the ImageJ software (NIH).

| Transwell assay
The transwell assay was performed to measure cell migration ability. The cells (2 × 10 4 /well in 200 μl DMEM) were plated in the upper chamber, and GCM (600 μl) was added to the lower chamber to stimulate cell migration. After 24 h, the remaining cells in the upper chamber were removed with a cotton swab, while the cells in the lower chamber were fixed with 4% PFA and stained with 0.1% crystal violet methanol. The number of migrated cells was quantified by counting the cells in six random microscopic fields (×100 magnification).

| Wound healing assay
HUVECs were seeded in six-well plates and allowed to reach a 90-100% confluence on the second day. Wounds were created with a 1.0-ml pipette tip by scratching straight lines on the cell surface, and detached cells were removed by washing with FBS-free DMEM. HUVECs were incubated with GCM for 48 h, wound images were captured under a light microscope (EVOS), and the wound area was analyzed using the ImageJ software.

| Tube forming assay
A precooled 24-well plate was coated with 50% Matrigel (100 μl /well) (Corning) and was left to polymerize at 37°C for 30 min. HUVECs (1 × 10 5 /well) were suspended in GCM supplemented with 10% FBS and seeded in 24-well plates for 12 h. Then, tube structures were photographed by a light microscope. Tubules were quantified and analyzed using the ImageJ software.

| Chick embryo chorioallantoic membrane (CAM) assay
The CAM assay was used to investigate angiogenesis effects following a previously described protocol. 18 Briefly, after incubation at 37°C for 8 days, a 1.0-cm window was created on the air sac of an egg (Linrui Breeding Co. Ltd) to expose the chorioallantoic membrane. A 0.5-cm diameter filter paper was first placed on top of the CAM, and 100 μl of GCM was added to the center of the paper. The window was covered with adhesive tape, and the eggs were incubated in a humid incubator at 37°C for 48 h. On day 10, blood vessels on the CAM were photographed with a digital camera (Canon). The numbers of secondand third-order vessels within the defined area of the membrane were manually counted to assess the effects of GCM on angiogenesis.

| Enzyme-linked immunosorbent assay (ELISA)
The concentration of VEGFA in the glioma cells medium was determined using an ELISA kit (Elabscience) according to the manufacturer's protocol. The absorbance was measured using a microplate reader at 450 nm.

| RNA isolation and quantitative real-time PCR assay
Total RNA was isolated from the cells and tissues by a TRIZOL kit (Invitrogen) based on the manufacturer's protocol. RNA was quantified and then reverse-transcribed into cDNA by a cDNA synthesis kit (Novoprotein). qRT-PCR was conducted by SYBR Green (Novoprotein) in an ABI 7000 thermal cycler. RNA expression was analyzed using the 2 −ΔΔCT method. GAPDH or U6 was used as the endogenous control for normalization. The primer sequences were synthesized by Tsingke Biotechnology. The sequence of the primers is outlined in Table S2.

| Western blot assay
The glioma cells were lysed using RIPA buffer with Protease and Phosphatase inhibitor (Beyotime). The total protein concentration was examined using a BCA kit. Equal amounts of total protein (30 μg) were electrophoresed in 10%-12% SDS-PAGE and electro-transferred onto NC membrane (GE). After blocking with 5% nonfat dry milk in TBST for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with HRP-labeled secondary antibodies (Proteintech) for 1 h at RT. An enhanced chemiluminescence reagent (Millipore) was used to detect protein expression value. The relative quantity of proteins was analyzed using the Image J software. The primary antibodies used are as follows

| RNA sequencing
Total RNA was isolated using an RNeasy kit (Qiagen) from three independent replicates of the U87 cells with SPRY4-IT1 knockdown or control. The RNA amount and purity of each sample were quantified. Further RNA sequencing and result analysis were executed by LC-Bio Technology Co., Ltd.
HE staining was performed with an HE kit (BaSO Biotechnology). The paraffin-embedded sections were sequentially deparaffinized, dehydrated, stained by hematoxylin, differentiated by the addition of hydrochloric ethanol, backed to blue with ammonia water, and stained with eosin. Afterward, the sections were dehydrated, cleared, and sealed. Finally, tissue HE photographs were acquired under a light microscope.

| Xenograft tumor assay
U87 shSPRY4-IT1 cells or scramble cells (3 × 10 5 in 10 μl PBS) were injected intracranially into female 6-week-old BALB/c nude mice (5 mice for each group). The mice were observed daily for death or neurological symptoms and were subsequently sacrificed when they exhibited neurological symptoms. Then, the whole brain was harvested, paraformaldehyde-fixed, paraffin-embedded, and sectioned coronally from anterior to posterior. The section with the largest tumor cross-sectional area was selected for tumor size measurement. The tumor volume was calculated using the formula: V = (a × b 2 )/2, where a and b represent the longest diameter and shortest diameter, respectively. Parameters a and b were measured using the Image J software.

| Bioinformatics analysis
Glioma TCGA datasets including SPRY4-IT1 expression values and patient survival were downloaded from the GlioVis website (https://gliov is.bioin fo.cnio.es/). The expression of SPRY4-IT1 and clinical data of glioma samples (WHO II: 194 samples, WHO III: 218 samples, WHO IV: 161 samples) were extracted and used for the survival analysis. The samples were divided into high and low groups according to the median SPRY4-IT1 expression value.

| Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8.0. Bars and error represent the mean ± standard deviation (mean ± SD) of replicate measurements. The means of normally distributed continuous data between two groups were analyzed using unpaired Student's t-tests.
One-way ANOVA was used to compare SPRY4-IT1 expression levels between different clinical grades of glioma. The Chi-square (χ 2 ) test was used to analyze the association between SPRY4-IT1 expression and clinicopathological characteristics of glioma. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Statistical significance was defined as p < 0.05.

| LncRNA SPRY4-IT1 expression is elevated in glioma and correlated with poor patient prognosis
To understand the expression profiles of lncRNA SPRY4-IT1 in glioma tissues and their prognostic significance, we first analyzed the TCGA sequencing data of glioma. The data demonstrated that SPRY4-IT1 mRNA increased along with WHO grades and was associated with poor outcomes in glioma patients ( Figure 1A,B). Subsequently, qRT-PCR was performed to detect SPRY4-IT1 expression in fresh glioma specimens with different WHO grades and non-brain tumors. The data revealed that SPRY4-IT1 expression was dramatically elevated in glioma tissues and gradually increased with WHO grades ( Figure 1C). In addition, we carried out a survival statistic (Kaplan-Meier analysis) to assess the relation between SPRY4-IT1 expression and survival time based on glioma patients' follow-up information. We found that glioma patients with higher SPRY4-IT1 levels had a shorter survival time ( Figure 1D). Further, according to the medium cutoff value of SPRY4-IT1 expression in glioma tissues, the patients were split into high and low expression groups. The glioma patients with a higher tumor grade exhibited higher SPRY4-IT1 expression (Table S1). Thus, these data suggest that SPRY4-IT1 overexpression could independently indicate poor prognosis in glioma patients.

| LncRNA SPRY4-IT1 promotes glioma cell proliferation in vitro
qRT-PCR analysis of SPRY4-IT1 mRNA levels in glioma cell lines and NHA showed that high expression values of SPRY4-IT1 were evident in most glioma cell lines (U87, U251, SNB19, and LN229) compared with NHA ( Figure 2A). It demonstrated that SPRY4-IT1 was highly expressed in glioma cell lines, in line with the observation in fresh glioma specimens. To verify the molecular functions of lncRNA SPRY4-IT1 in glioma cells, U87, SNB19, and U118 cells were chosen for subsequent experiments.
First, we silenced SPRY4-IT1 expression using siRNA transfection in U87 and SNB19 cells, or overexpressed SPRY4-IT1 by pcDNA3.1 plasmid transfection in U118 cells. qRT-PCR was used to analyze the efficiency of knockdown or overexpression ( Figure 2B). Next, a CCK-8 assay was conducted to evaluate cell proliferation viability. The data revealed that silencing of SPRY4-IT1 suppressed U87 and SNB19 cells' growth, and overexpression of SPRY4-IT1 enhanced U118 cells' proliferation ( Figure 2C,D). Then, an EdU assay was conducted to further examine the effect of SPRY4-IT1 on cell proliferation. Consistently, the data indicated more EdU-positive cells when SPRY4-IT1 was overexpressed, and the inhibition of SPRY4-IT1 significantly decreased the number of EdUpositive cells ( Figure 2E,F). Collectively, these data indicate that SPRY4-IT1 could induce glioma cell proliferation in vitro.

| LncRNA SPRY4-IT1 enhances the ability of glioma cells to induce angiogenesis
To further investigate the molecular mechanism of how SPRY4-IT1 promoted glioma cell proliferation, RNAsequencing was performed to compare the transcriptomes of U87 siSPRY4-IT1-NC and U87 siSPRY4-IT1 cells. The Volcano plot demonstrated that 739 genes were upregulated and 508 genes were downregulated, compared with U87 siSPRY4-IT1-NC cells (>2 folds, p < 0.05, one-way ANOVA test) ( Figure 3A). Based on these differentially expressed genes, GO enrichment analysis revealed that the extracellular matrix organization, cell migration regulation and, angiogenesis were significantly enriched ( Figure 3B). Previous studies have confirmed that SPRY4-IT1 could regulate cell proliferation and migration in various tumors, 19,20 including glioma. 15 However, whether lncRNA SPRY4-IT1 could regulate tumor angiogenesis is yet unknown. Thus, we performed glioma cell-mediated vascular endothelial cell tubule formation assays. The results revealed that the culture medium (CM) from SPRY4-IT1 silenced U87 and SNB19 cells significantly induced less tubule formation of HUVECs compared with the control group, while CM of SPRY4-IT1 overexpressed U118 cells strongly stimulated more tubule formation of HUVECs than the control group ( Figure 3C,D). Meanwhile, to investigate the effect of SPRY4-IT1 on angiogenesis in vivo, we conducted a CAM assay. Treatment with CM from the SPRY4-IT1-silenced U87 and SNB19 cells decreased the vessel number compared with the control group, while CM from SPRY4-IT1-overexpressed U118 cells promoted the formation of more vessels than the control group ( Figure 3E,F). Therefore, these results indicate that SPRY4-IT1 enhanced glioma cell-mediated angiogenesis.

| LncRNA SPRY4-IT1 promotes the ability of glioma cells to induce proliferation and migration of HUVECs
Here, we investigated the potential role of lncRNA SPRY4-IT1 in endothelial cell proliferative and migrative functions, which are critical for angiogenesis. CM of SPRY4-IT1 silenced U87 and SNB19 cells effectively suppressed the proliferation of HUVECs, while CM of SPRY4-IT1-overexpressed U118 cells promoted the proliferation of HUVECs (Figure 4A,B). Then, we evaluated the effect of CM on HUVECs migration by transwell assay, and the data revealed that CM derived from SPRY4-IT1-knockdown U87 and SNB19 cells could weaken the migration of HUVECs. In contrast, CM from SPRY4-IT1-overexpressed U118 cells stimulated the migration of HUVECs ( Figure 4C,D). Consistent with the transwell assay, similar results were observed for the scratch wound assay. As depicted in Figure 4E, the results demonstrated that CM from SPRY4-IT1-knockdown glioma cells significantly decreased HUVECs migration distance, and CM from SPRY4-IT1-overexpressed glioma cells markedly increased HUVECs migration distance ( Figure 4E,F). These findings demonstrated that SPRY4-IT1 has a key role in inducing endothelial cell proliferation and migration in glioma cells.

| LncRNA SPRY4-IT1 regulates EZH2 and VEGFA expression in glioma cells
We further analyzed data of RNA-sequencing and identified 23 genes (upregulated 10 genes; downregulated 13 genes) that had the most significant changes in transcription level in a heatmap ( Figure 5A). Among the genes with the most significant changes, we were more interested in EZH2, a well-known tumor gene that drives the malignant progression of various tumors. Next, we selected all the significant genes under the angiogenesis GO item and analyzed their correlation with EZH2 by  Figure 5B, the correlation heatmap displayed that only VEGFA and EZH2 were correlated, and the correlation coefficient reached 0.94. Furthermore, immunoblotting analysis was performed to detect whether the expression of EZH2 and VEGFA are regulated by SPRY4-IT1 in glioma cells. The data revealed that SPRY4-IT1 could significantly upregulate EZH2 and VEGFA ( Figure 5C). Moreover, ELISA results indicated that VEGFA protein levels were downregulated and upregulated in SPRY4-IT1-silenced and -overexpressed glioma cells, respectively, compared to the control cells ( Figure 5D). These data suggest that SPRY4-IT1 could promote the cell proliferation and angiogenesis of glioma by inducing the expression of EZH2 and VEGFA.

| LncRNA SPRY4-IT1 promotes glioma cell proliferation and angiogenesis via the miR-101-3p/EZH2/VEGFA signaling axis
Here, we performed rescue experiments to investigate whether the miR-101-3p/EZH2/VEGFA axis, involved in the lncRNA SPRY4-IT1, promoted glioma cells proliferation and induced angiogenesis. siSPRY4-IT1 and EZH2 overexpression plasmid were co-transfected into U87 and SNB19 cells, then CCK-8 and EdU tests were performed. The results showed that this co-transfection partially reversed the proliferation of glioma cells compared with siS-PRY4-IT1 cells ( Figure 7A-D). CAM and HUVECs tube formation assays showed that this co-transfection largely rescued the ability of glioma cells to induce angiogenesis ( Figure 7E-H). Next, a VEGFA-neutralizing antibody was combined with the GCM obtained from SPRY4-IT1overexpressed U118 cells. CCK-8 results showed that compared with SPRY4-IT1-overexpressed U118 GCM treatment alone, the cell viability of HUVECs was decreased after treatment with the VEGFA-neutralizing antibody ( Figure 8A,B). Similar results were observed on transwell and scratch wound assays, which showed that the cell migration capability of HUVECs was decreased when combined with VEGFA-neutralizing antibody treatment compared with SPRY4-IT1-overexpressed U118 GCM treatment alone ( Figure 8C-F). HUVECs tube formation and CAM assays demonstrated that the angiogenesis effects induced by SPRY4-IT1-overexpressed U118 GCM were weakened when combined with VEGFAneutralizing antibody treatment ( Figure 8G-J). Therefore, the rescue experiment results demonstrated that the effect of SPRY4-IT1 on glioma cell proliferation and angiogenesis occurred partly through the miR-101-3p/EZH2/ VEGFA axis.

| LncRNA SPRY4-IT1 function confirmed in orthotopic nude mice model
To investigate the relevance of lncRNA SPRY4-IT1 loss in glioma cells in vivo, we established an U87-shSPRY4-IT1 stable cell line. U87-shSPRY4-IT1 cells and U87-scramble cells were orthotopically xenografted by intracerebral injection into immunocompromised mice. As expected, HE staining demonstrated that the tumor size of the U87-shSPRY4-IT1 mice was decreased compared to the scramble mice ( Figure 9A,B). Moreover, KM survival analysis demonstrated that the survival time of the mice was noticeably prolonged after silencing SPRY4-IT1 ( Figure 9C). Further, immunohistochemistry demonstrated that the expression of EZH2, VEGFA, and CD31 in the in situ tumors of shSPRY4-IT1 mice were significantly reduced ( Figure 9D). The above experiments confirmed that lncRNA SPRY4-IT1 promoted glioma growth and induced glioma angiogenesis in vivo. A schematic diagram displaying the SPRY4-IT1/miR-101-3p/ EZH2/VEGFA signaling axis promoting glioma tumorigenesis and angiogenesis is presented in Figure 9E.

| DISCUSSION
In this study, we provided clinical evidence that lncRNA SPRY4-IT1 was highly expressed in glioma tissues and was associated with poor patient outcomes. We further elucidated that SPRY4-IT1 promotes glioma cell proliferation and angiogenesis in vitro and in vivo, and activates the miR-101-3p/EZH2/VEGFA pathway. These findings indicate that SPRY4-IT1 induced glioma tumorigenesis and angiogenesis, and could be a promising prognostic indicator in patients with glioma.
LncRNAs have been regarded as important mediators in glioma development. 21 Here, we investigated the possible involvement of lncRNA SPRY4-IT1 in glioma progression. Although previous reports have also identified that SPRY4-IT1 was upregulated in glioma specimens and was related with poor outcomes in patients with glioma, 14 F I G U R E 6 LncRNA SPRY4-IT1 regulates VEGFA expression in glioma cells via the miR-101-3p/EZH2 signaling axis. (A) The predicted binding site between SPRY4-IT1 and miR-101-3p and the putative binding site between miR-101-3p and EZH2 3'UTR. (B) miR-101-3p expression was detected by qRT-PCR in glioma cells with SPRY4-IT1-silenced or SPRY4-IT1-overexpressed. (C) EZH2 and VEGFA protein levels were determined by immunoblot in U87 and SNB19 cells with EZH2-silenced. (D) EZH2 and VEGFA protein levels were analyzed by immunoblot in U87, SNB19 cells co-transfected with siSPRY4-IT1 and miR-101-3p inhibitor. (E) EZH2 and VEGFA protein levels were analyzed by immunoblot in U87 and SNB19 cells co-transfected with siSPRY4-IT1 and EZH2 overexpression plasmid. (F) The expression correlation between SPRY4-IT1, EZH2, and VEGFA of patients with glioma from the TCGA dataset. *p < 0.05, **p < 0.01 and ***p < 0.001. reported that knockdown of SPRY4-IT1 suppressed U251 and SF295 cell proliferation. 15,22 In this current study, U87 and SNB19 cells with relatively high SPRY4-IT1 expression and U118 cells with relatively low SPRY4-IT1 expression were chosen for our research. We found that SPRY4-IT1 promoted glioma cell proliferation in a series of functional experiments both in vitro and in vivo.
To further elucidate the molecular mechanism of SPRY4-IT1 promoting the growth of glioma cells, we performed RNA-sequencing to compare the transcriptomes of U87 siSPRY4-IT1-NC and U87 siSPRY4-IT1 cells. GO enrichment analysis results revealed that the term of angiogenesis was significantly enriched. Inducing angiogenesis is one of the most crucial hallmarks of cancer, and anti-angiogenic therapy has recently become one of the key strategies in anti-tumor therapy. 23 Angiogenesis is a universal characteristic of glioma progression that facilitates proliferation and migration. 24 Bevacizumab, a humanized monoclonal antibody against VEGF, was approved as a clinical first-line chemotherapeutic drug to treat recurrent glioblastoma. 25 Therefore, antiangiogenesis therapy is essential to limit the progression of tumors, including gliomas. Current evidence suggests that lncRNA could exert a modulatory role in glioma angiogenesis, such as H19, XIST, HULC, and PAXIP1-AS1. [26][27][28][29] However, the relationship between SPRY4-IT1 and glioma angiogenesis remained unknown. In this study, we observed that a conditioned medium of glioma cells with knockdown or overexpressed SPRY4-IT1 could suppress or stimulate the tube formation of HUVECs in vitro and angiogenesis of CAM in vivo, respectively. Our study provides evidence that SPRY4-IT1 functions as an angiogenesis regulator in glioma. Besides, the clinical glioma specimen's immunohistochemistry results also demonstrated a positive correlation between SPRY4-IT1 value and vascular endothelial marker (CD34).
To further investigate the downstream mechanism of EZH2, we analyzed the correlation between EZH2 and differential genes under the angiogenesis GO item using the Spearman analysis. The results showed that only VEGFA and EZH2 were relevant. VEGFA, a crucial molecular of tumor angiogenesis, plays a critical role in endothelial cell proliferation, migration, and tube formation, 36 and has also been reported to promote cell cycle, proliferation, and metastasis in glioma as an oncogene. 37 In our study, we found that the expression of VEGFA was stably downregulated in SPRY4-IT1-silenced glioma cells, while VEGFA was strongly upregulated in SPRY4-IT1overexpressed cells. Consistently, the VEGFA concentration in the conditioned medium from SPRY4-IT1-silenced or SPRY4-IT1-overexpressed glioma cells was decreased and increased, respectively. Liu et al. reported that EZH2 contributed to the progression of lung adenocarcinoma by elevating the expression of VEGFA. 38 Geng et al. also reported that EZH2 could promote non-small cell lung cancer progression by upregulating the VEGFA/AKT signaling axis. 39 Our data demonstrated that VEGFA expression was significantly downregulated following EZH2 inhibition, and VEGFA protein levels were restored in glioma cells after co-transfection with siSPRY4-IT1 and EZH2-overexpression plasmid, suggesting that SPRY4-IT1 regulated VEGFA expression through the mediation of EZH2 in glioma cells. VEGFA, an effective angiogenic factor, binds to VEGFR2 and activates downstream effect pathways to promote the proliferation and migration of endothelial cells. Sun et al. reported that VEGF could induce VEGFR2 endocytosis and downstream AKT/ ERK signaling activation in HUVECs. 40 Consistent with Sun's report, our results showed that phospho-VEGFR2, phospho-AKT, and phospho-ERK1/2 expression levels were downregulated in HUVECs treatment in a conditioned medium from SPRY4-IT1-silenced glioma cells, which significantly weakened HUVECs proliferation and migration viability. Collectively, these data indicated that SPRY4-IT1 promoted glioma cell proliferation and angiogenesis via the miR-101-3p/EZH2 axis by mediating VEGFA downregulation.
In the current clinical treatments of glioma patients, bevacizumab (anti-VEGF monoclonal antibody) is used as a first-line chemotherapeutic drug for anti-angiogenic therapy. Many clinical trials suggested that the clinical prognosis of glioma patients was improved with combined treatment using Temozolomide (TMZ) and bevacizumab. 41,42 The efficacy of combining lentivirus-shSPRY4-IT1 and TMZ (or bevacizumab, or TMZ and bevacizumab) in inhibiting glioma cells or xenograft glioma model should also be investigated. More research is still required to fully elucidate the significance and potential targeting of SPRY4-IT1.
In summary, the present studies indicate that lncRNA SPRY4-IT1 regulates VEGFA expression to facilitate cell proliferation and angiogenesis of glioma via the miR-101-3p/EZH2 axis, and our findings provide mechanistic insights into potential strategies for anti-angiogenic therapy in glioma.