Knockdown of long non-coding RNA AGAP2-AS1 suppresses the proliferation and metastasis of glioma by targeting microRNA-497-5p

ABSTRACT

Long non-coding RNA (LncRNA) AGAP2-AS1 has been demonstrated to involve in various malignancies. However, the expression and biological effect of AGAP2-AS1 on glioma remain enigmatic. We aimed to explore the effects of AGAP2-AS1 on glioma. Expressions and relationship of AGAP2-AS1 and microRNA-497-5p (miR-497-5p) in different grades of glioma tissues and cell lines as well as normal ones were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), starBase, and dual-luciferase reporter assay. In addition, the effect of AGAP2-AS1 on the cell biological behaviors, epithelial-mesenchymal transition (EMT)-related markers, and miR-497-5p level was detected by cell functional experiments, western blot, and qRT-PCR. After transfection with miR-497-5p mimic (M), inhibitor (I), and AGAP2-AS1 knockdown, miR-497-5p level, cell biological behaviors, and EMT-related markers were detected again. AGAP2-AS1 expression was increased while miR-497-5p expression was decreased in glioma tissues and cell lines, and increase of AGAP2-AS1 expression or reduction of miR-497-5p expression was also correlated with clinicopathological grades of glioma. Furthermore, AGAP2-AS1 knockdown repressed cell biological behaviors and EMT-related markers expressions. Mechanically, AGAP2-AS1 targeted miR-497-5p and AGAP2-AS1 knockdown led to elevation of miR-497-5p expression. In addition, rescue experiments were conducted to validate the vital influence of miR-497-5p on the AGAP2-AS1-regulated proliferation and metastasis of glioma. AGAP2-AS1 may serve as an oncogene in the tumorigenesis of glioma by inhibiting miR-497-5p expression, showing its potential as a prognostic biomarker and a therapeutic target for glioma.

KEYWORDS:

• Glioma
• LncRNA AGAP2-AS1
• MiRNA-497-5p
• proliferation
• metastasis

View publisher note:

Publisher’s Note

Introduction

Glioma is one of the most aggressive malignant tumors, which has a high incidence and mortality in the world [1,2]. In clinical practice, glioma has developed into advanced stage when diagnosed, and the prognosis of glioma patients is poor [3]. In the past decades, progress has been made in surgery, radiotherapy and chemotherapy. However, the 5-year survival rate remains very low [4]. Therefore, it is of great urgency to pay more attention to the effective diagnosis and treatment of glioma, and explore the internal mechanism of the occurrence and progression of glioma.

A complex gene interaction has been reported to participate in the development of glioma and molecularly targeted therapies are essential for meliorating glioma prognosis [5,6]. Recently, more and more non-coding genes have been discovered. Long non-coding RNAs (LncRNAs) are a type of RNAs with a transcription length of over 200 nucleotides. Accumulating researches have manifested that lncRNAs play extremely vital roles in the occurrence and progression of malignant tumors, such as cell differentiation, proliferation, metastasis, as well as apoptosis, etc. [7–11]. Growing evidence has proved that lncRNAs are abnormally expressed and perform important roles in glioma. For instance, lncRNA PLAC2 inhibits cell cycle progression in glioma by down-regulating RPL36 expression [5]. LncRNA UCA1 has been reported to interact with miR-182 and regulate glioma progression by targeting iASPP [12]. Zhang et al have confirmed that lncRNA CCND2-AS1 represses cell proliferation through Wnt/beta-catenin pathway in glioma [13]. LncRNA MALAT1/miR-129 axis accelerates glioma tumorigenesis by targeting SRY (sex determining region Y)-box 2 (SOX2) [14]. Besides, the ectopic expression of lncRNA AGAP2-AS1 has been confirmed to be closely associated with glioma [15], but its mechanism is still unclear.

MicroRNAs (miRNAs, miRs) are group of small non-coding RNAs including about 22 nucleotides [16–18]. MiRNAs interfere with the structural stability and translation of the mRNA by binding to the complementary sequence of target mRNA at 3ʹ-untranslated region (3ʹ-UTR) [19,20]. There is ample evidence showing that miRNAs play key roles in multiple diseases by regulating related cell functions [21–23]. For instance, in gastric cancer, lncRNA HOTAIR modulates the gastric cancer progression through miR-331-3p/human epithelial growth factor receptor 2 (HER2) [24]. LncRNAH19 affects the proliferation of hepatocellular carcinoma cell by mediating miR-675 [25]. Besides, lncRNA NEAT1 regulates the functions of colorectal cancer cell through miR-150-5p/cleavage and polyadenylation specific factor 4 (CPSF4) axis [26].

Based on the above evidence, we have reason to hypothesize that the AGAP2-AS1/miRNA axis may affect the progression of glioma. The purpose of our present study was to probe the effect and mechanism of AGAP2-AS1 and its downstream target miRNA on the proliferation and invasion of glioma cell. We discovered that deletion of AGAP2-AS1 restrained the proliferation and invasion of glioma cell, providing both experimental basis for the molecular mechanism of glioma development and a novel target for the treatment of glioma. Altogether, this research exhibited the function of a novel ceRNA pathway in glioma.

Materials and methods

Tissue specimens

A total of 60 pairs of glioma and normal tissue specimens were collected from patients without any therapeutic intervention from May 2018 to June 2019 in the First Affiliated Hospital of Nanjing Medical University (2,018,030,026). The differentiation status was graded according to the method proposed by Edmondson and Steine. Written informed consent was received from all patients. Tissue specimens were immediately stored at −80°C.

Cell culture and transfection

The glioma cell lines, including U251 (C1135), T98G (C1372), A172 (C1203), and normal human astrocyte cell line (HEB, C1246) were bought from WHELAB (Shanghai, China). A172 and U251 cells were cultivated in complete medium (M0101, WHELAB, China). HEB and T98G cells were cultivated in complete medium (M0301, WHELAB, China). LN229 (CL-0578) and SHG44 (CL-0207), another glioma cell lines, were obtained from Procell (Wuhan, China) and maintained in DMEM (PM150210, Procell, China) supplemented with 10% fetal bovine serum (FBS, 164,210, Procell, China) and 1% penicillin-streptomycin solution (PB180120, Procell, China). All cells were placed in an incubator (51,030,999, Thermo Scientific, USA) at 37°C with 5% CO_2.

Small interfering RNA targeting AGAP2-AS1 (siAGAP2-AS1, sequence: 5ʹ-CACTTGTTACCTGCTTTATAAAT-3ʹ) was constructed from YouBio (China) and inserted into the Silencer 4.1-CMV neo vector (VT1395, YouBio, China). Empty vector was used as negative control (NC). MiR-497-5p inhibitor (I, miR20002820-1-5, sequence: 5ʹ-ACAAACCACAGUGUGCUGCUG-3ʹ), inhibitor control (IC, miR2N0000001-1-5, sequence: 5ʹ-CAGUACUUUUGUGUAGUACAA-3ʹ), miR-497-5p mimic (M, miR10002820-1-5, sequence: 5ʹ-CAGCAGCACACUGUGGUUUGUAAACCAC AGUGUGCUGCUGUU-3ʹ), and mimic control (MC, miR1N0000001-1-5, sequence: 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ) were got from RiboBio (Guangzhou, China). Then, we used Lipofectamine 2000 (12,566,014, Invitrogen, USA) to transfect the above plasmids into glioma cells. After 48 hours (h), relative expression patterns of AGAP2-AS1 and miR-497-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR).

Cell processing

First, we concentrated on the effect of AGAP2-AS1 on glioma cells, the cells were divided into the Blank group (Cells were cultured normally), siNC group (Cells were transfected with siNC), and siAGAP2-AS1 group (Cells were transfected with siAGAP2-AS1). Next, we focused on the effects of AGAP2-AS1 and miR-497-5p on cells, the cells were divided into the MC/M group (Cells were transfected with miR-497-5p MC or M), IC/I group (Cells were transfected with miR-497-5p IC or I), and siAGAP2-AS1 + I group (Cells were transfected with siAGAP2-AS1 and miR-497-5p I).

QRT-PCR

Trizol reagent (abs60031, absin, China) was employed to separate total RNA from frozen glioma tissues and cell lines following the manufacturer’s protocol. The RNA was reversely transcribed to cDNA using the 1st Strand cDNA Synthesis SuperMix (abs60077, absin, China) or miRNA cDNA first-strand synthesis kit (N126, Jiancheng, China). QRT-PCR experiments were conducted using SYBR High-Sensitivity qPCR SuperMix (abs60086, absin, China) on the PCR system (ABI7900, Applied Biosystems, USA). GAPDH or U6 was employed as housekeeping genes and the specific primers were listed in Table 1. The 2^−ΔΔCT method was used to analyze the gene expression levels [27].

Table 1. Gene sequence primers.

Name	forward	reverse
AGAP2-AS1	5ʹ-TACCTTGACCTTGCTGCTCTC-3’	5ʹ-TGTCCCTTAATGACCCCATCC-3’
miR-497-5p	5′-TGCGGCAGCAGCACACTGTG-3′	5′-TGCGGCAGCAGCACACTGTG-3′
U6	5ʹ-CTCGCTTCGGCAGCACA-3’	5ʹ-AACGCTTCACGAATTTGCGT-3’
GAPDH	5′-ACCTGACCTGCCGTCTAGAA-3′	5′-TCCACCACCTGTTGCTGTA-3′

Cell Counting Kit-8 (CCK-8) assay

Cell viability was measured using CCK-8 kit (C0037, Beyotime, China). Transfected cells (1 × 10⁴) were added in a 96-well plate and grown for different time points (0, 24, 48 and 72 h). Subsequently, 10 μL CCK-8 reagents were added to each well and incubated at 37°C for another 4 h. Thereafter, the absorbance was examined at 450 nm using a microplate reader (Z742711, Sigma, USA) [28].

Colony formation assay

The colony formation assay was applied to measure the cell proliferation ability. In brief, transfected cells (8 × 10²) were inoculated in a 96-well plate and grown for two weeks until visible cell clones appeared. Cells were washed and fixed in methanol (M140298, Aladdin, China), and then stained by Giemsa dye solution (C0133, Beyotime, China). Subsequently, the number of visible colonies was counted using the optical microscope (Z723975-1EA, Sigma, USA) [29].

Wound healing assay

Wound-healing assay was applied to investigate the functions of AGAP2-AS1 and miR-497-5p in glioma cell migration. Briefly, treated cells at a density with 1 × 10⁵cell/mL were grown in a 6-well plate for 24 h. Then, the pipette tip was employed to make a linear scratch. Following that the cells were cultured for 48 h, their scratch gaps were observed and photographed under a light microscope (100×) at 0 and 48 h, respectively [30].

Transwell chamber assay

In brief, glioma cells (1 × 10⁴) were re-suspended in a medium without FBS and seeded to the upper chamber pre-coated with Matrigel. Then, the lower chamber was added with complete medium. After being probed for 48 h, the invaded cells were fixed with methanol and stained with 0.1% crystal violet (C0121, Beyotime, China). At last, these cells were quantified using the optical microscope (200×) in five randomly chosen microscopic fields [31].

Western blot analysis

RIPA buffer (abs9229, absin, China) was used to extract total protein from cells and the Bicinchoninic acid (BCA) assay kit (abs9232, absin, China) was applied to analyze the concentration of proteins [32]. Protein samples were separated by 10% SDS-PAGE and transferred to the nitrocellulose membranes (2215, Millipore, USA). Subsequently, we used 5% nonfat milk to block the proteins at 37°C for 2 h. The membranes were then incubated with primary antibodies against E-cadherin (1:1000, 135 kDa, #14,472, Cell Signaling Technology, USA), N-cadherin (1:1000, 140 kDa, #14,215, Cell Signaling Technology, USA), Snail (1:1000, 29 kDa, #3895, Cell Signaling Technology, USA), and GAPDH (1:1000, 36 kDa, #97,166, Cell Signaling Technology, USA) at 4°C overnight. After another 1 h of incubation with goat anti-mouse secondary antibody (1:5000, ab190475, Abcam, UK) at 37°C, protein bands were visualized by the ECL reagent (abs920, absin, China) under a gel imaging system (A44114, Invitrogen, USA). Proteins were normalized to the GAPDH.

Dual-luciferase reporter assay

The complementary binding site of AGAP2-AS1 and miR-497-5p were predicted through starBase (http://starbase.sysu.edu.cn/index.php). The 3ʹ-UTR fragment, containing the binding site of miR-497-5p, was inserted into the pmirGLO vector for the construction of AGAP2-AS1-wild type (WT) plasmid and the corresponding AGAP2-AS1-mutant (MUT) was harvested from Ribobio in Guangdong, China. Cells transfection was conducted using Lipofectamine 2000. After 48 h, the luciferase activities were analyzed using the dual-luciferase reporter assay system (GloMax 2020, Promega, USA) [31].

Statistical analysis

Data were presented as mean ± standard deviation. All experiments were carried out at least three times and statistical analysis was performed using SPSS 21.0 software (IBM Corp.). The levels of AGAP2-AS1 and miR-497-5p in 60 glioma tissues and 60 adjacent non-cancer tissues were analyzed by paired-samples t test. An independent sample t test was utilized to examine the differences between two groups. Multiple comparisons were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. P< 0.05 was considered as statistically significant.

Results

LncRNA AGAP2-AS1 has been demonstrated to involve in various malignancies. However, the expression and biological effect of AGAP2-AS1 on glioma remain enigmatic. The purpose of our present study was to probe the effect and mechanism of AGAP2-AS1 and its downstream target miRNA on the glioma cell biological behaviors and EMT process. We discovered that AGAP2-AS1 was overexpressed in glioma tissues and cells. Depletion of AGAP2-AS1 repressed biological behaviors and EMT process of glioma cells. We further confirmed that AGAP2-AS1 could target miR-497-5p and miR-497-5p expression was decreased in glioma tissues and cells. AGAP2-AS1 facilitated the biological behaviors and EMT process of glioma cells by targeting miR-497-5p.

LncRNA AGAP2-AS1 expression was increased in glioma tissues and cell lines

Firstly, we assessed the AGAP2-AS1 expression in tissue specimens and cell lines to unveil the biological function of AGAP2-AS1 in glioma. The results of qRT-PCR assay indicated that AGAP2-AS1 expression was enhanced in the glioma tissue specimens compared with that in the normal tissues (Figure 1(a), p< 0.01). The expression of AGAP2-AS1 in glioma tissues with WHO II–IV grade was higher than that in glioma tissues with WHO I grade, and the increase in WHO grade was positively correlated with AGAP2-AS1 expression (Figure 1(b), p< 0.01). As shown in Figure 1(c), we discovered the expression of AGAP2-AS1 in glioma cell lines was notably up-regulated compared with that in the HEB cells (P< 0.01). As the AGAP2-AS1 expression was the highest in T98G cells and the lowest in U251 cells, T98G and U251 cells were selected for follow-up experiments.

Figure 1. LncRNA AGAP2-AS1 was highly expressed in glioma tissues and cell lines. (a) AGAP2-AS1 expression in 60 glioma tissues and 60 normal samples was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. **P< 0.01 vs. Normal group. (b) AGAP2-AS1 expression in different grades of glioma was quantified by qRT-PCR (WHO I group, n = 6; WHO II group, n = 19; WHO III group, n = 21; WHO IV group, n = 14). GAPDH was served as the internal control. **P< 0.01 vs. WHO I group; ^##P< 0.01 vs. WHO II group. (c) The expression of AGAP2-AS1 in glioma cell lines and normal human astrocyte cell line (HEB) was measured by qRT-PCR, with GAPDH as the internal control. **P< 0.01 vs. HEB group. All experiments were performed in triplicate and data were expressed as mean ± standard deviation (SD).

Inhibition of AGAP2-AS1 attenuated the viability and proliferation of glioma cells

To explore the pathological role of AGAP2-AS1 in the modulation of glioma function, siAGAP2-AS1 and siNC were transfected into U251 and T98G cells. The transfection efficiency was validated and the data indicated that the AGAP2-AS1 expression was markedly decreased in U251 and T98G cells transfected with siAGAP2-AS1 (Figure 2(a–b), P< 0.01). Functionally, CCK-8 assay signified that siAGAP2-AS1 dramatically repressed the viability of U251 and T98G cells (Figure 2(c–d), P< 0.01). Consistently, colony formation assay demonstrated that AGAP2-AS1 inhibition notably restrained the proliferation ability of glioma cells (Figure 2(e–f), P< 0.01).

Figure 2. Knockdown of AGAP2-AS1 inhibited proliferation of glioma cells. (a-b) The expression of AGAP2-AS1 in U251 and T98G cells transfected with small interfering RNA targeting AGAP2-AS1 (siAGAP2-AS1) or its negative control (siNC) was evaluated by qRT-PCR assay. GAPDH was utilized as the internal control. (c-d) The viability of U251 and T98G cells was determined by Cell Counting Kit-8 (CCK-8) assay. (e-f) The proliferation of U251 and T98G cells was determined by colony formation assay. **P< 0.01 vs. siNC group. All experiments were performed in triplicate and data were presented as mean ± SD.

Down-regulation of AGAP2-AS1 repressed migration and invasion abilities of glioma cells

We performed wound healing and transwell assays to investigate the cell migration and invasion. Wound healing assay suggested that the migration abilities of U251 and T98G cells were remarkably restrained in the siAGAP2-AS1 group when compared with those in the siNC group (Figure 3(a–b), P< 0.01). The results of the transwell assay showed that the deletion of AGAP2-AS1 obviously inhibited the invasion of glioma cells (Figure 3(c–d), P< 0.01).

Figure 3. Knockdown of AGAP2-AS1 repressed migration and invasion of glioma cells. (a-b) The migration abilities in U251 and T98G cells were examined by wound healing assay (magnification 100×, scale bar = 100 µm). (c, d) The invasion abilities in U251 and T98G cells after transfection were evaluated by transwell assay (magnification 200×, scale bar = 50 µm). **P< 0.01 vs. siNC group. All experiments were performed in triplicate and data were expressed as mean ± SD.

Knockdown of AGAP2-AS1 restrained the expressions of EMT-related markers in glioma cells

At the molecular level, we performed western blot method to identify the expression patterns of EMT-associated markers. The expression of E-cadherin was elevated, while those of N-cadherin and Snail were decreased after knockdown of AGAP2-AS1 in U251 and T98G cells (Figure 4(a–b), P< 0.01).

Figure 4. Knockdown of AGAP2-AS1 restrained epithelial-mesenchymal transition (EMT) of glioma cells. (a-b) Western blot was conducted to detect the expressions of EMT-related markers, with GAPDH as the internal control. **P< 0.01 vs. siNC group. All experiments were performed in triplicate and data were expressed as mean ± SD.

AGAP2-AS1 targeted miR-497-5p and downregulation of miR-497-5p was evidenced in glioma tissues and cells

To the best of our knowledge, lncRNAs have multiple biological functions through sponging various miRNAs. To dissect the molecular target of AGAP2-AS1 in glioma, starBase database has been utilized to screen the miRNAs and identify that miR-497-5p may be the potential target of AGAP2-AS1 (Figure 5(a)). We found that miR-497-5p M extremely repressed the luciferase activity of AGAP2-AS1-WT (Figure 5(b–c), P< 0.01), but no prominent changes were viewed in that of AGAP2-AS1-MUT. Next, we further confirmed that miR-497-5p expression was declined in the glioma tissue specimens compared with that in the normal tissues (Figure 5(d), p< 0.01). The expression of miR-497-5p in glioma tissues with WHO II–IV grade was lower than that in glioma tissues with WHO I grade and the increase in WHO grade was negatively correlated with miR-497-5p expression (Figure 5(e), p< 0.01). Meanwhile, the data presented that miR-497-5p expression in glioma cell lines was notably restrained compared with that in the HEB cells (figure 5(f), p< 0.01). The qRT-PCR assay manifested that the expression of miR-497-5p was increased by siAGAP2-AS1 (Figure 5(g–h), P< 0.01).

Figure 5. AGAP2-AS1 targeted miR-497-5p and downregulation of miR-497-5p was observed in glioma tissues and cells. (a) The binding sites between AGAP2-AS1 and miR-497-5p were predicted by starBase (http://starbase.sysu.edu.cn/index.php). (b-c) To estimate the interaction between AGAP2-AS1 and miR-497-5p, dual-luciferase reporter assay was implemented. **P< 0.01 vs. Blank group. (d) MiR-497-5p expression in 60 glioma tissues and 60 normal samples was assessed by qRT-PCR assay. U6 was utilized as the internal control. **P< 0.01 vs. Normal group. (e) MiR-497-5p expression in different grades of glioma was quantified by qRT-PCR (WHO I group, n = 6; WHO II group, n = 19; WHO III group, n = 21; WHO IV group, n = 14). U6 acted as the internal control. **P< 0.01 vs. WHO I group; ^##P< 0.01 vs. WHO II group. (f) MiR-497-5p expression in glioma cell lines and HEB cells was measured by qRT-PCR, with U6 as the internal control. **P< 0.01 vs. HEB group. (g-h) MiR-497-5p expression in glioma cells transfected with siAGAP2-AS1 or siNC was evaluated by qRT-PCR assay. U6 was used as the internal control. **P< 0.01 vs. siNC group. All experiments were performed in triplicate and data were expressed as mean ± SD.

AGAP2-AS1 facilitated the glioma cell biological behaviors by targeting miR-497-5p

As displayed in Figure 6(a–b), qRT-PCR proved that the miR-497-5p expression was up-regulated in U251 and T98G cells transfected with miR-497-5p M, while the opposite trend was observed in cells transfected with miR-497-5p I (P< 0.01). Moreover, siAGAP2-AS1 overturned the inhibition of miR-497-5p I on miR-497-5p expression (Figure 6(a–b), P< 0.01). Next, we performed the rescue experiment to investigate the connection between AGAP2-AS1 and miR-497-5p in glioma. The data proved that miR-497-5p M reduced the cell viability and proliferation, while miR-497-5p I ran conversely (Figure 6(c–d), P< 0.01). Co-transfection of siAGAP2-AS1 and miR-497-5p I partially offset the promotive effect of miR-497-5p I on the cell viability and proliferation (Figure 6(c–d), P< 0.01). Consistent with these results, down-regulation of AGAP2-AS1 counteracted the promotive effect of miR-497-5p I on the migration and invasion of U251 and T98G cells (Figure 7(a–d), P< 0.01).

Figure 6. AGAP2-AS1 facilitated the glioma cell viability and proliferation by targeting miR-497-5p. (a-b) U251 and T98G cells were transfected with miR-497-5p mimic (m), miR-497-5p inhibitor (i) or siAGAP2-AS1 and the expression level of miR-497-5p in U251 and T98G cells was determined by qRT-PCR assay. U6 was served as the internal control. (c-f) The viability and proliferation of U251 and T98G cells after transfection were assessed by CCK-8 and colony formation assays. **P< 0.01 vs. MC group, ^##P< 0.01 vs. IC group, ^{^^}P< 0.01 vs. I group. All experiments were performed in triplicate and data were expressed as mean ± SD.

Figure 7. AGAP2-AS1 facilitated the glioma cell migration and invasion by targeting miR-497-5p. (a-b) U251 and T98G cells were transfected with miR-497-5p M, miR-497-5p I or siAGAP2-AS1 and the migration abilities of U251 and T98G cells were examined by wound healing assay (magnification 100×, scale bar = 100 µm). (c-d) The invasion abilities of U251 and T98G cells were evaluated by transwell assay after transfection (magnification 200×, scale bar = 50 µm). **P< 0.01 vs. MC group, ^##P< 0.01 vs. IC group, ^{^^}P< 0.01 vs. I group. All experiments were performed in triplicate and data were expressed as mean ± SD.

AGAP2-AS1 facilitated the expressions of EMT-related markers in glioma cells by targeting miR-497-5p

In Figure 8(a–b), the western blot assay manifested that miR-497-5p I decreased the expression of E-cadherin and increased the expressions of N-cadherin and Snail in glioma cells, while the effect as such was reversed by siAGAP2-AS1 (P< 0.01).

Figure 8. AGAP2-AS1 facilitated the expressions of EMT-related markers in glioma cells by targeting miR-497-5p. (a-b) The regulatory effects of AGAP2-AS1 and miR-497-5p on the EMT-related markers were determined by western blot after miR-497-5p M, miR-497-5p I or siAGAP2-AS1 was transfected into U251 and T98G cells. **P< 0.01 vs. MC group, ^##P< 0.01 vs. IC group, ^{^^}P< 0.01 vs. I group. All experiments were performed in triplicate and data were expressed as mean ± SD.

Discussion

Currently, various diagnostic and therapeutic strategies have been applied in glioma [33,34]. However, the overall survival rate of glioma remains unsatisfactory. Numbers of studies have demonstrated that lncRNAs are important regulators for a variety of molecular and cellular activities, affecting numerous physiological and pathological processes [35–37]. Particularly, the involvement of lncRNAs in regulating glioma progression has been widely confirmed [38–40].

Recent studies have indicated that lncRNA AGAP2-AS1 is abnormally expressed in many cancers. For example, lncRNA AGAP2-AS1 accelerates colorectal cancer progression via regulating miR-4668-3p/SRSF1 axis [41]. Additionally, lncRNA AGAP2-AS1 is an independent predictor in renal carcinoma patients [42]. Knockdown of lncRNA AGAP2-AS1 represses the proliferation through miR-195-5p/FOSL1 axis [43]. Zheng et al have confirmed that AGAP2-AS1 level is augmented in glioma tissues and cells, and AGAP2-AS1 intensified the expression of HDGF by adsorbing miR-15a/b-5p to facilitate the malignant progression of glioma [44]. In line with previous research, the results of our study confirmed that AGAP2-AS1 was overexpressed in glioma tissue specimens and cell lines, demonstrating that AGAP2-AS1 may be acted as a prognostic biomarker for the patients of glioma. Functionally, AGAP2-AS1 knockdown suppressed the biological behaviors of glioma cells.

Growing evidence has demonstrated that lncRNAs regulate post-transcriptional translation of genes through targeting miRNAs, mRNAs, as well as proteins, thus exerting a pivotal effect in the occurrence and development of various cancers [45]. Increasing researches have shown that miR-497 [46], as a tumor suppressor, blocks the growth of miscellaneous cancers. For example, one research has manifested that miR-497 represses the growth of osteosarcoma tumor by targeting plexinA4 and CDK6 [47]. Another study clarified that lncRNA DLX6-AS1 regulated YAP1 expression by adsorbing miR-497-5p to promote the proliferation, migration and invasion of neuroblastoma [48]. Cheng et al have elucidated that lncRNA LINC00662/miR-497-5p/EglN2 axis aggravates the proliferation and metastasis of breast cancer cells [49]. Additionally, an existing research has underlined that ELFN1-AS1 augments the proliferation, migration, and invasion of ovarian cancer cells via targeting miR-497-3p [31]. Our study proved for the first time that miR-497-5p expression was decreased in glioma tissues and cells, and the increase in WHO grade was negatively correlated with miR-497-5p expression, supplementing the deficiency of miR-497-5p in glioma.

In order to further evaluate the relationship between AGAP2-AS1 and miR-497-5p, we identified the binding sites of miR-497-5p and AGAP2-AS1. Furthermore, we determined the vital sites reliable for the usable interaction with miR-497-5p and found that AGAP2-AS1 functioned as a sponge for the miR-497-5p. Nevertheless, to clarify whether miR-497-5p mediated the function of AGAP2-AS1 on the development of glioma, the rescue experiments were performed and the results revealed that miR-497-5p I facilitated the viability, migration and invasion of glioma cells. However, those trends were reversed by co-transfection of siAGAP2-AS1 and miR-497-5p I.

Next, to further investigate the mechanism via which the AGAP2-AS1/miR-497-5p axis regulated the biological behaviors of glioma cells, we dug into the phenotype-associated genes, and discovered that the migration and invasion of glioma cells were closely related to EMT [50]. Thereinto, one of the main markers of EMT was E-cadherin, and the down-regulation of E-cadherin led to a decrease in the instability of cell adhesion, while the up-regulation of the mesenchymal molecular marker N-cadherin induced the migration of tumor cells [51,52]. In addition, there were many other transcription factors that regulated gene expression, which also helped to suppress epithelial phenotype and activate mesenchymal phenotype, including Snail [53]. One prior study reported that there were enriched EMT-related genes in glioma cells and revealed that EMT played a pivotal role in glioma [54]. The complex interaction of lncRNA-miRNA-mRNA may be the underlying cause of EMT progression in glioma [2]. Our research further manifested that miR-497-5p I decreased the expression of E-cadherin and increased the expressions of N-cadherin and Snail in glioma cells, while siAGAP2-AS1 offset the regulation of miR-497-5p I on the expressions of EMT-related markers. There are still some deficiencies in this study. Whether AGAP2-AS1 targets miRNAs other than miR-497-5p affects the progression of glioma. We will combine multiple databases to predict miRNAs that target AGAP2-AS1 and miRNAs that are abnormally expressed in glioma. Next, we made a Venn diagram to obtain miRNAs. Moreover, another limitation of this study is that the effect of AGAP2-AS1 on glioma was confirmed only at the cellular level. In the future, we will conduct animal experiments to further confirm the results of this study.

Conclusion

In summary, we revealed that AGAP2-AS1 was overexpressed and was related to the grade of glioma. Specifically, our findings illuminated that down-regulation of AGAP2-AS1 restrained cell proliferation and invasion and EMT in glioma by targeting miR-497-5p. The excavation of novel molecular biomarkers for glioma may contribute to improve the level of clinical treatment. Collectively, we elucidated the roles and specific mechanism of AGAP2-AS1 in glioma progression, thereby providing a deep understanding of AGAP2-AS1’s function in glioma and developing AGAP2-AS1 as a prognostic biomarker for glioma patients.

Highlight

1. AGAP2-AS1 was overexpressed in glioma tissues and cells.

2. AGAP2-AS1 silencing repressed cell biological behaviors and EMT process.

3. MiR-497-5p was lower expressed in glioma tissues and cells.

4. AGAP2-AS1 facilitated the progression of glioma by targeting miR-497-5p.

Availability of Data and Materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors’ contributions

Substantial contributions to conception and design: Yi Sun

Data acquisition, data analysis and interpretation: Yulong Shen, Xing Li

Drafting the article or critically revising it for important intellectual content: Yi Sun

Final approval of the version to be published: Yi Sun, Yulong Shen, Xing Li

Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved: Yi Sun, Yulong Shen, Xing Li

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.