Abstract

The present study was undertaken to investigate the underlying mechanisms of long noncoding RNA OIP5-AS1 via regulating miR-410 to modulate Wnt-7b in the progression of glioma. To address this problem, we measured the expression of OIP5-AS1 and miR-410 in glioma tissues by qRT-PCR. Glioma U87 cells were transfected with OIP5-AS1 siRNA or miR-410 inhibitors. The targeting relationships among miR-410, OIP5-AS1 and Wnt-7b were verified by luciferase reporter assays. Western blotting was employed to determine the expression of Wnt-7b/β-catenin pathway-related proteins, while MTT, flow cytometry, Transwell assays and wound-healing assays were used to measure the biological characteristics of glioma cells. The results showed that OIP5-AS1 expression was higher and miR-410 was lower in glioma tissues. Luciferase reporter assays confirmed a targeting relationship between OIP5-AS1 and miR-410, as well as between miR-410 and Wnt-7b. Silencing OIP5-AS1 reduced cell proliferation, invasion and migration of glioma U87 cells and led to depressed expression levels of miR-410, Wnt-7b, p-β-catenin, GSK-3β-pS9, c-Myc and cyclin D1. Furthermore, down-regulation of OIP5-AS1 induced G0/G1 phase cell cycle arrest and apoptosis of glioma cells. Inhibitors of miR-410 abolished the biological effects of OIP5-AS1 siRNA in glioma cells. In vivo, OIP5-AS1 knockdown also inhibited tumor growth. Taken together, this research suggested that silencing OIP5-AS1 may specifically block the Wnt-7b/β-catenin pathway via targeted up-regulating miR-410, thereby inhibiting growth, invasion and migration while promoting apoptosis in glioma cells.

Introduction

Glioma, as a very common malignant tumor in the human central nervous system, features rapid growth, strong invasion, high mortality and high disability rates, accounting for approximately 60% of adult primary brain tumors [1,2]. Although great advances have been made in comprehensive therapies of glioma, including radiotherapy, chemotherapy and surgery, the clinical management of glioma still faces many challenges. In particular, glioma is usually found in hidden areas of the brain and is diagnosed at advanced/late stages, possibly causing difficulties for thorough resection and displaying resistance to both chemotherapy and radiotherapy [3,4]. The average survival time of patients diagnosed with glioma was only approximately 14 months with a 5-year survival rate of no more than 5% [5]. Therefore, it is necessary to study glioma progression at the molecular level and to identify new therapeutic approaches [6]. Currently, with the deepening understanding and application of molecular targeted therapy for glioma, the role of long noncoding RNA (lncRNA) in glioma development has attracted clinical attention [7]. LncRNAs, consisting of more than 200 nucleotides without protein-coding potential [8], have been suggested to be substantially involved in the development and progression of glioma. For example, lncRNA TUG1 was identified as a tumor suppressor by Li et al. [9] to be down-regulated in glioma tissues, and it has the potential to be applied for the treatment of glioma. Another group reported that overexpressed lncRNA H19 in glioma tissues was negatively linked to the glioma prognosis and that silencing H19 inhibited the growth of glioma cells by modulating miR-675 [10].

OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1), a member of the lncRNA family, is located on the human chromosome 15q15.1 and is evolutionarily conserved in vertebrates [11]. According to the study by Kim et al. [12], OIP5-AS1, a competing endogenous RNA (ceRNA) for HuR, suppressed the growth of the cervical cancer cell line HeLa. Yang et al. [13] also showed that OIP5-AS1 regulated the expression of miR-410 and modulated its target gene KLF10, thus affecting the downstream PTEN/PI3K/AKT signaling pathway in multiple myeloma by facilitating cell proliferation, cycle progression and apoptosis inhibition. Meanwhile, as illustrated by the study of Chen et al. [14], miR-410 acts on its target gene MET to effectively regulate the proliferation and invasion of glioma cells. Actually, miR-410 has been proven to specifically inhibit Wnt-7b expression, thereby influencing the proliferation and invasion of oral squamous cell carcinoma cells [15]. Furthermore, OIP5-AS1 is important for controlling neurogenesis during development, because it is highly expressed in the nervous system, as well as some types of human cancers [16]. Limited data are available concerning the possible association between OIP5-AS1 and glioma; however, Liu et al. [17] reported that PIWIL3/piR-30188/OIP5-AS1 regulated the biological behavior of glioma cells via the miR-367-3p/CEBPA/TRAF4 pathway. Therefore, after identifying targeting relationships between OIP5-AS1 and miR-410 and between miR-410 and Wnt-7b using a biological information prediction website, we investigated whether OIP5-AS1 regulated miR-410 to target Wnt-7b and affect glioma development.

Materials and methods

Ethics statement

The study obtained approval of the Ethics Committee of The Second Hospital of Hebei Medical University and informed consent was obtained from all patients prior to the study. Furthermore, the animal experiments were approved by the Ethics Committee for Laboratory Animals, and all procedures were performed strictly in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH) [18].

Study subjects

From October 2016 to October 2017, tissue samples were obtained from 65 glioma patients who underwent surgical resection at the Department of Neurosurgery in our hospital; diagnoses were confirmed by the pathological examination. Hematoxylin–eosin (HE) staining was used to observe glioma morphology (Figure 1A). Patients had not received any other therapies prior to surgery, and complications such as other malignant tumors, serious systemic infections and other severe systemic diseases were ruled out. The patients had a mean age of 46.23 ± 12.58 years, including 40 males and 25 females. Based on the pathological classification of tumors of the central nervous system published by WHO in 2007 [19], there were 29 cases of low-grade glioma (grade I/II), including 12 cases of grade I and 17 cases of grade II, and 36 cases of high-grade glioma (grade III/IV), including 19 cases of grade III and 17 cases of grade IV. Non-neoplastic brain tissue samples were obtained from 10 adult patients with craniocerebral injuries who underwent partial resections of brain tissues to reduce intracranial pressure. All samples were preserved at –80°C for subsequent experiments.

Expression of OIP5-AS1 and miR-410 in glioma tissues and normal tissues measured by qRT-PCR

Figure 1
Expression of OIP5-AS1 and miR-410 in glioma tissues and normal tissues measured by qRT-PCR

(A) HE staining used to observe glioma morphology. (B) The relative expression of OIP5-AS1 and miR-410 in glioma tissues and normal tissues. *P<0.05 compared with normal tissues. (C) The correlation analysis between expression levels of OIP5-AS1 and miR-410, P<0.05 indicating a statistically significant difference; (D and E) Relative expression levels of OIP5-AS1 (D) and miR-410 (E) in various grades of glioma tissues; *P<0.05 compared with the low grade glioma tissues.

Figure 1
Expression of OIP5-AS1 and miR-410 in glioma tissues and normal tissues measured by qRT-PCR

(A) HE staining used to observe glioma morphology. (B) The relative expression of OIP5-AS1 and miR-410 in glioma tissues and normal tissues. *P<0.05 compared with normal tissues. (C) The correlation analysis between expression levels of OIP5-AS1 and miR-410, P<0.05 indicating a statistically significant difference; (D and E) Relative expression levels of OIP5-AS1 (D) and miR-410 (E) in various grades of glioma tissues; *P<0.05 compared with the low grade glioma tissues.

Culture and transfection of glioma cells

The human U87 glioma cell line was provided by the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (HyClone Company, U.S.A.) and 1% streptomycin (Promega Company, U.S.A.), with the conditions of 5% CO2, 37°C and 95% humidity. OIP5-AS1 siRNA, negative control (NC) sequences and miR-410 inhibitors were synthesized by Shanghai GenePharma Co., Ltd. The sequence of the OIP5-AS1-targeting siRNA was 5′-GCAGCAUGCUGUGUGCAAA-3′, the negative control (NC) sequence was 5′-GAAATGTACTTGAGCGTGGAGAC-3′ and the sequence of miR-410 inhibitors was 5′-UUAUAUUGUGUCUACCGGACA-3′. Cells were assigned into five groups: Blank group (cells without any transfection), NC group (cells transfected with NC sequence), OIP5-AS1 siRNA group (cells transfected with OIP5-AS1 siRNA), miR-410 inhibitors group (cells transfected with miR-410 inhibitors) and OIP5-AS1 siRNA+miR-410 inhibitors (siRNA+ inhibitors) group (cells transfected with OIP5-AS1 siRNA and miR-410 inhibitors).

First, U87 cells collected during the logarithmic growth phase were digested, centrifuged, made into single cell suspensions with antibiotic-free medium, and seeded into six-well plates at 2 × 105 cells/well before 12–24 h of incubation. Cells were cultured in complete medium at least 24 h before transfection. A total of 100 nM of OIP5-AS1 siRNA, siRNA NC or miR-410 inhibitors were diluted separately in 200 μl of Opti-MEM medium. After incubation for 15 min at room temperature, the oligonucleotides were added, and the cells were incubated for 48 h. When cells were adherent to the bottom and walls of the plate and cell confluence reached 50–70%. Lipofectamine-2000 (Invitrogen) was utilized for the transfection of U87 cells according to the manufacturer’s protocol.

Dual-luciferase reporter assay

OIP5-AS1 and wnt-7b 3′-UTR fragments containing the binding site of miR-410 were amplified to construct wild-type plasmids OIP5-AS1-wt and Wnt-7b-wt, expressing the binding sites of miR-410 to OIP5-AS1 and wnt-7b, respectively, as well as mutant type plasmids OIP5-AS1-mt and Wnt-7b-mt, expressing the binding sites of miR-410 to OIP5-AS1 and wnt-7b, respectively. During the experiment, the human embryonic kidney cell line HEK 293T cells (purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were obtained at the logarithmic growth phase, which were seeded to the 24-well plate and cultured in the DMEM medium with 10% FBS. When the cell confluence rose to 80%, cells were transfected by following the manual of the Lipofectamine 2000 (Invitrogen) reagent kit. Approximately 48 h after transfection, a dual-luciferase reporter gene assay kit (Promega) was used for detection before calculating the ratio of the firefly luciferase activity to Renilla luciferase activity. The experiment was performed three times to obtain mean values.

Expression levels of OIP5-AS1, miR-410 and Wnt-7b measured by qRT-PCR

The TRIzol reagent kit (Invitrogen, U.S.A.) was employed for the extraction of total RNA from glioma tissues, normal tissues and cells in each group; primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table 1). Next, total RNA (2 μg) was reverse-transcribed into cDNA with a Reverse Transcription Kit (Takara, Biochemical, Tokyo, Japan). In the subsequent step, the cDNA was amplified using a SYBR Premix Ex Taq II (Perfect Real-Time) kit (Akara, Biochemical, Tokyo, Japan). U6 was the internal reference gene for miR-410 and GAPDH was used for OIP5-AS1. The 2−ΔΔCt method was applied for the calculation of the relative expression of target genes. Each experiment was repeated three times independently.

Table 1
Primer sequences for qRT-PCR
Target gene Primer sequences 
OIP5-AS1 Forward: 5′-TGCGAAGATGGCGGAGTAAG-3′ 
 Reverse: 5′-TAGTTCCTCTCCTCTGGCCG-3′ 
miR-410 Forward: 5′-AGTTGTTCACCACCTTCTCCAC-3′ 
 Reverse: 5′-TATCGTTGTACTCCAGTCCAAGTC-3′ 
U6 Forward: 5′-GTGCTCGCTTCGGCAGCACATATAC-3′ 
 Reverse: 5′-AAAAATATGGAACGCTCACGAATTTG-3′ 
GAPDH Forward: 5′-CATGAGAAGTATGACAACAGCCT-3′ 
 Reverse: 5′-AGTCCTTCCACGATACCAAAGT-3′ 
Target gene Primer sequences 
OIP5-AS1 Forward: 5′-TGCGAAGATGGCGGAGTAAG-3′ 
 Reverse: 5′-TAGTTCCTCTCCTCTGGCCG-3′ 
miR-410 Forward: 5′-AGTTGTTCACCACCTTCTCCAC-3′ 
 Reverse: 5′-TATCGTTGTACTCCAGTCCAAGTC-3′ 
U6 Forward: 5′-GTGCTCGCTTCGGCAGCACATATAC-3′ 
 Reverse: 5′-AAAAATATGGAACGCTCACGAATTTG-3′ 
GAPDH Forward: 5′-CATGAGAAGTATGACAACAGCCT-3′ 
 Reverse: 5′-AGTCCTTCCACGATACCAAAGT-3′ 

Western blotting analysis

Cells were disrupted by RIPA lysis and the concentration of proteins was calculated by determination with a bicinchoninic acid (BCA) protein assay (Thermo, U.S.A.). Next, protein separation was achieved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred onto nitrocellulose membranes. Then, the membranes were placed in a refrigerator for overnight reaction at 4°C with primary antibodies, as follows: Wnt-7b (Batch No. ab10297, Abcam, U.S.A.), β-catenin (Batch No. ab32572, Abcam, U.S.A.), GSK-3β (Batch No. ab93926, Abcam, U.S.A.), c-Myc (1:1000 diluted, Batch No. ab32072, Abcam, U.S.A.), Cyclin D1 (Batch No. ab134175, Abcam, U.S.A.) and GAPDH (Batch No. ab9485, Abcam, U.S.A.). Later, secondary antibodies were added for 1 h of incubation at room temperature, followed by chemiluminescence, development, fixation and result analysis. These experiments were performed three times to obtain mean values.

Cell proliferation assessed by MTT assay

Cells collected at the logarithmic growth phase were digested with trypsin and adjusted for cell density until it was 1 × 105 cells/ml. At 24, 48, 72 and 96 h after the cells were seeded in 96-well plates, 20 μl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (Sigma, U.S.A.) solution (5 mg/ml) was added to each well for 4 h of incubation. Next, the solution in each well was removed, and 150 μl of dimethyl sulfoxide (DMSO) solution (Sigma, U.S.A.) was added into each well for 10 min of oscillation. Finally, an enzyme-linked immunosorbent assay (ELISA) reader (BIO-RAD, Cal, U.S.A.) was employed to measure the OD (optical density) value of each well under the wavelength of 490 nm. To obtain the mean value, the experiment was repeated three times independently.

Cell invasion detected by Transwell assay

After overnight thawing, Matrigel was diluted by DMEM medium three times its own volume and plated into the upper chambers of Transwell systems. Approximately 2 × 104 cells were inoculated into each well of the upper chamber, while 0.5 ml of DMEM medium containing 10% FBS was added to each well of the lower chamber simultaneously. After incubation for 24 h, the cells were fixed with paraformaldehyde and stained for half an hour with 0.1% Crystal Violet. Next, cells that did not go through the upper chamber were washed away with 0.1 ml of phosphate buffer saline (PBS). Finally, an optical microscope was used for the observation, counting and imaging. The experiment was repeated three times and the mean values were obtained.

Cell migration measured by wound-healing assay

A marker pen was used to draw three parallel lines on the back of the 12-well plate, and then 1 × 104 cells were seeded to each well. When cells evenly covered each well, a pipette tip was employed to scratch lines vertical to the three lines on the back of the plate, and 0.1 ml of PBS buffer was used to wash away the shed cells. An optical microscope was used for the observation and imaging of cells at 0 and 48 h of incubation in the culture medium. The experiment was repeated three times to obtain mean values.

Cell cycle and apoptosis evaluated by flow cytometry

After transfection for 48 h, cells were collected, digested with 0.25% trypsin and adjusted to the density of 1 × 106 cells/ml. Next, 1 ml of cell suspensions were centrifuged for 10 min at the rate of 1500 rpm; the supernatants were removed, and 2 ml of PBS buffer was added for another round of centrifugation. Then, the supernatants were discarded, and precooled 70% ethanol was added for the overnight fixation of cells at 4°C. On the next day, 50 μg of RNAase-containing propidium iodide (PI) was added into 100 μl of cell suspension for 30 min of reaction without exposure to light, filtered by a 100-mesh nylon filter screen. A flow cytometer (BD, FL, NJ, U.S.A.) was used to measure the cell cycle (under the excitation wavelength of 488 nm), and Annexin V-FITC/ PI staining was used to detect the apoptosis of cells. Subsequently, cells were collected, washed twice with PBS buffer, centrifuged and suspended in 200 μl of binding buffer. Next, 10 μl of Annexin V-FITC (ab14085, Abcam, Inc, MA, U.S.A.) and 5 μl of PI were added for 15 min of reaction at room temperature without exposure to light, and then 300 μl of binding buffer was added. Finally, a flow cytometer was employed to detect apoptosis under the excitation wavelength of 488 nm. The experiment was repeated three times to obtain mean values.

Tumorigenicity in nude mice

Thirty-two male BALB/c nude mice were provided by Hunan SJA Laboratory Animal Co., Ltd. (license no. SCXK [Xiang] 2009-0009) and were assigned into four groups with eight per group. Mice were injected subcutaneously with cell suspensions (approximately 5 × 106 cells/100 μl) in the Blank, NC, OIP5-AS1 siRNA and miR-410 inhibitors groups. After formation of tumors, both length (a) and width (b) of the tumor bodies were measured with Vernier calipers, and tumor volume was calculated based on the formula: V = (ab2)/2. The growth curves of tumors were drawn accordingly. Three weeks after tumor inoculation, the mice were killed by cervical dislocation. Tumor tissues were obtained, and tumor weights were measured. The tumor tissues obtained from mice were then stained with HE.

Statistical analysis

All data were processed and analyzed by using the statistics software SPSS 20.0 (SPSS, Inc, Chicago, U.S.A.). Measurement data were presented by mean ± standard deviation (). Mean values between two groups were compared using independent samples t-tests, while mean values among several groups analyzed by one-way ANOVA. Intergroup comparisons were tested by the least significant difference-test (LSD-t), and correlation analysis was conducted using the Pearson correlation coefficient. P<0.05 was regarded as statistically significant.

Results

Expression of OIP5-AS1 and miR-410 in glioma tissues

The expression levels of OIP5-AS1 and miR-410 in glioma tissues and normal tissues were evaluated by qRT-PCR, and the data in human tissues showed that the expression levels of OIP5-AS1 were up-regulated, but the expression levels of miR-410 were down-regulated in glioma tissues compared with normal tissues (both P<0.05, Figure 1B). According to the correlation analysis results, expression levels of OIP5-AS1 negatively correlated to levels of miR-410 (r = −0.645, P<0.05, Figure 1C). Furthermore, expression levels of OIP5-AS1 and miR-410 were significantly associated with the pathological grading of glioma, because OIP5-AS1 was notably more highly expressed in high-grade glioma tissues than in low-grade tissues (P<0.05, Figure 1D); however, miR-410 levels were lower in high-grade glioma than in low-grade tissues (P<0.05, Figure 1E).

The targeting relationship among miR-410, OIP5-AS1 and Wnt-7b

OIP5-AS1 is a molecular sponge that regulates miR-410, as predicted by Starbase 2.0. With the use of online prediction software Target Scan, we identified the binding site between Wnt-7b and miR-410 (Figure 2A,B). According to the dual-luciferase activity assay, miR-410 mimics did not significantly differ from the NC group with respect to the effect on the luciferase activity of OIP5-AS1-mt and Wnt-7b-mt (all P>0.05); however, miR-410 mimics significantly down-regulated the luciferase activity of wild-type OIP5-AS1-wt and Wnt-7b-wt (all P<0.05, Figure 2C,D). These results suggested that there was a targeting relationship between OIP5-AS1 and miR-410, as well as between miR-410 and Wnt-7b.

The targeting relationship among miR-410, OIP5-AS1 and Wnt-7b

Figure 2
The targeting relationship among miR-410, OIP5-AS1 and Wnt-7b

(A and B) The online bioinformatics software predicted the binding sites of miR-410 to OIP5-AS1 and Wnt-7b. (C and D) The targeting relationship between miR-410 and OIP5-AS1 and between miR-410 and Wnt-7b verified by the dual-luciferase reporter assay; *P<0.05 compared with the NC group.

Figure 2
The targeting relationship among miR-410, OIP5-AS1 and Wnt-7b

(A and B) The online bioinformatics software predicted the binding sites of miR-410 to OIP5-AS1 and Wnt-7b. (C and D) The targeting relationship between miR-410 and OIP5-AS1 and between miR-410 and Wnt-7b verified by the dual-luciferase reporter assay; *P<0.05 compared with the NC group.

Expression of Wnt-7b/β-catenin signaling pathway-related proteins

We then examined the protein expression levels in U87 glioma cells and found that there was no difference between the NC and Blank groups with respect to expression levels of each protein, and the expression levels of total GSK-3β and total β-catenin were not significantly different in all groups (all P>0.05). Furthermore, protein expression levels of Wnt-7b, p-β-catenin, GSK-3β-pS9, c-Myc and cyclin D1 were lower in the OIP5-AS1 siRNA group (all P<0.05) but were remarkably higher in the miR-410 inhibitors group compared with the Blank group (all P<0.05). Moreover, cells in the siRNA+ inhibitors group were dramatically lower in terms of expression of miR-410 and increased in terms of expression of Wnt-7b/β-catenin pathway-related proteins when compared with cells in the OIP5-AS1 siRNA group (Figure 3, all P<0.05).

Expression of miR-410 and Wnt-7b/β-catenin signaling pathway-related proteins in each group

Figure 3
Expression of miR-410 and Wnt-7b/β-catenin signaling pathway-related proteins in each group

(A) The expression of Wnt-7b/β-catenin signaling pathway-related proteins assessed by Western blotting. (B) Comparison of the expression of miR-410 in each group. (CG), Comparison of the expression of Wnt-7b/β-catenin signaling pathway-related proteins in each group; *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Figure 3
Expression of miR-410 and Wnt-7b/β-catenin signaling pathway-related proteins in each group

(A) The expression of Wnt-7b/β-catenin signaling pathway-related proteins assessed by Western blotting. (B) Comparison of the expression of miR-410 in each group. (CG), Comparison of the expression of Wnt-7b/β-catenin signaling pathway-related proteins in each group; *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Comparison of cell proliferation among different groups

Proliferation of U87 glioma cells in each group was evaluated using the MTT assay (Figure 4). First, there was no significant difference between the Blank and NC groups with respect to proliferation at various time points (P>0.05). Proliferation was apparently inhibited in the OIP5-AS1 siRNA group and was remarkably enhanced in the miR-410 inhibitors group compared with cells in the Blank group (all P<0.05). On the other hand, in comparison with the OIP5-AS1 siRNA group, the siRNA+ inhibitors group increased dramatically in terms of proliferation (all P<0.05), suggesting that miR-410 inhibitors reversed the inhibitory effect of OIP5-AS1 siRNA on proliferation.

Proliferation of cells in various groups at various time points detected by MTT assay

Figure 4
Proliferation of cells in various groups at various time points detected by MTT assay
Figure 4
Proliferation of cells in various groups at various time points detected by MTT assay

Cell cycle and cell apoptosis of different groups

We further investigated the mechanism of transfection of the OIP5-AS1 on cell cycle and apoptosis in U87 human glioma cells. There were no differences between the NC and Blank groups with respect to the proportion of G0/G1 phase, S phase and G2 phase (all P>0.05). Compared with cells in the Blank group, those in the OIP5-AS1 siRNA group showed a significantly elevated proportion of G0/G1 phase, a reduced proportion of S phase, and apparently increased apoptosis rates (all P<0.05, Figure 5). Cells in the miR-410 inhibitors group were significantly reduced in terms of proportion of G0/G1 phase cells and apoptosis rates, with elevated proportions of S phase cells (all P<0.05). On the other hand, by comparison with cells in the OIP5-AS1 siRNA group, those in the siRNA+ inhibitors group showed substantially fewer cells in the G0/G1 phase and lower apoptosis rates (all P<0.05), with an increased proportion of S phase cells (all P<0.05). No significant differences were found in terms of the proportion of G2 phase in all groups (all P>0.05).

Comparison of cell cycle and apoptosis rates among groups

Figure 5
Comparison of cell cycle and apoptosis rates among groups

Cell cycle (A) and apoptosis (B) among groups detected by flow cytometry. Comparison of cell cycle (C) and apoptosis rates (D) among groups. *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Figure 5
Comparison of cell cycle and apoptosis rates among groups

Cell cycle (A) and apoptosis (B) among groups detected by flow cytometry. Comparison of cell cycle (C) and apoptosis rates (D) among groups. *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Comparison of cell invasion and migration among different groups

Transwell and wound-healing assays were employed to assess invasion and migration of U87 human glioma cells, respectively. As shown in Figure 6, the NC group showed no observable difference from the Blank group in terms of the number of invasive cells and the wound-healing rate (P>0.05). However, the number of invasive cells and the wound-healing rate were substantially lower in the OIP5-AS1 siRNA group and significantly higher in the miR-410 inhibitors group (all P<0.05). Furthermore, the siRNA+ inhibitors group was statistically higher than the OIP5-AS1 siRNA group with respect to these two indexes (all P<0.05).

Comparison of cell invasion and migration among groups

Figure 6
Comparison of cell invasion and migration among groups

Cell invasion and migration in each group detected by Transwell (A) and wound-healing assays (B). Comparison of the number of invasive cells (C) and wound-healing rate (D) among groups. *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Figure 6
Comparison of cell invasion and migration among groups

Cell invasion and migration in each group detected by Transwell (A) and wound-healing assays (B). Comparison of the number of invasive cells (C) and wound-healing rate (D) among groups. *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group; &P<0.05 compared with the miR-410 inhibitors group.

Effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivo

As demonstrated by the experiment of tumorigenicity in an in vivo nude mice model, the nude mice in the NC group were not significantly different in terms of tumor volume or weight at various time points compared with those of the Blank group (all P>0.05). For the nude mice in the OIP5-AS1 siRNA group, their tumor growth was inhibited with significantly reduced tumor volume and weight when compared with mice in the Blank and NC groups (all P<0.05); the miR-410 inhibitors group was completely opposite to the OIP5-AS1 siRNA group in terms of the changes in these indexes (all P<0.05, Figure 7A,B). HE staining of tumors in the Blank and NC groups showed typical histological features of glioma, including high cellular density and an infiltrative growth pattern; the changes were substantial in miR-410 inhibitors group. In the OIP5-AS1 siRNA group, necrotic foci were observed, tissue alignment became irregular, and glioma cellular density decreased significantly compared with the other four groups (Figure 7C).

Effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivo

Figure 7
Effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivo

(A) The tumor volume growth curve of the nude mice in each group (n=8). (B) The subcutaneous tumor weight of the nude mice in each group (n=8); *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group. (C) Representative HE staining of the subcutaneous glioma in different groups was shown.

Figure 7
Effect of OIP5-AS1 and miR-410 on the tumorigenicity of glioma cells in vivo

(A) The tumor volume growth curve of the nude mice in each group (n=8). (B) The subcutaneous tumor weight of the nude mice in each group (n=8); *P<0.05 compared with the Blank group; #P<0.05 compared with the OIP5-AS1 siRNA group. (C) Representative HE staining of the subcutaneous glioma in different groups was shown.

Discussion

We found that OIP5-AS1 was significantly increased and miR-410 was decreased in glioma tissues, displaying a negative correlation relationship between OIP5-AS1 and miR-410 expression levels. As documented previously, OIP5-AS1 may act as a ceRNA to combine with HuR, thereby inhibiting the expression of HuR to participate in the phenotypic regulation of tumor cells [12], and lncRNA is increasingly identified as expressed aberrantly in tumors. Similarly, a study by Deng et al. [20] reported that OIP5-AS1 exerted oncogenic roles when highly expressed in lung adenocarcinoma tissues and cells via competitively binding to miR-448 to affect proliferation, migration and invasion. Furthermore, miR-410 was down-regulated in human glioma tissues, acting as a tumor suppressor to greatly affect glioma growth by down-regulating its target gene MET [14]. Therefore, we hypothesized that OIP5-AS1 may act as an oncogene and miR-410 may act as a tumor suppressor in glioma. We also found that the expression of OIP5-AS1 and miR-410 had a good deal to do with the pathological grading of glioma. To be specific, higher pathological gradings of glioma patients demonstrated higher expression levels of OIP5-AS1 and lower expression levels of miR-410, suggesting that OIP5-AS1 and miR-410 were closely related to the pathogenesis and development of glioma.

To investigate the regulation of OIP5-AS1 and miR-410 on the growth of glioma, we chose U87 glioma cells for transfection in vitro. We found that silencing OIP5-AS1 using siRNAs in U87 glioma cells inhibited cell growth via effectively suppressing proliferation, invasion and migration capabilities, and promoting apoptosis, as well as inducing G0/G1 phase cell cycle arrest. Consistent with our finding, Naemura et al. [11] also reported that silencing of OIP5-AS1 modulated the cell cycle and thereby regulated the proliferation of cervical cancer HeLa cells. In the present study, the trend of the miR-410 inhibitors group was observed to be completely opposite to that of the OIP5-AS1 siRNA group, suggesting that blocking miR-410 reversed the inhibitory role of OIP5-AS1 siRNA in terms of growth and metastasis. More importantly, our dual-luciferase assay confirmed the targeting relationship between OIP5-AS1 and miR-410, suggesting that OIP5-AS1 may play roles in glioma pathogenesis and progression by modulation of miR-410.

Expression of Wnt-7b/β-catenin signaling pathway-related proteins was determined to further elucidate the underlying mechanism of OIP5-AS1 in glioma. Expression levels of Wnt-7b, p-β-catenin, GSK-3β-pS9, c-Myc and Cyclin D1 were dramatically down-regulated and the expression of miR-410 was up-regulated in the OIP5-AS1 siRNA group. Nevertheless, cells in the miR-410 inhibitors group showed a completely opposite trend in the changes of these indexes to the OIP5-AS1 siRNA group, showing that inhibition of miR-410 reversed the effect of OIP5-AS1 siRNA to activate the activity of the Wnt-7b/β-catenin pathway. Similarly, as illustrated by Shiah et al. [15], miR-410 attenuated the Wnt-β-catenin pathway in oral squamous cell carcinoma cells by targeting Wnt-7b, an activator of the Wnt-β-catenin pathway. Furthermore, Wnt-7b levels were markedly lower in glioma tissues than in nontumor tissues, as illustrated Zhang et al [21]. Notably, the Wnt7b signaling pathway was shown to regulate distinct glioma–vascular interactions and tumor microenvironments [22]. Using the dual-luciferase reporter assay, we also confirmed that Wnt-7b was indeed the target gene of miR-410, suggesting that silencing OIP5-AS1 may affect growth and metastasis of U87 glioma cells via targeted regulation of Wnt-7b by miR-410. Wnt-7b serves as an important agonist of the Wnt/β-catenin signaling pathway [23,24], possibly preventing the phosphorylation and degradation of β-catenin induced by GSK-3β inhibition in cytoplasm; the accumulated β-catenin would translocate to the nucleus to bind with T-cell factor/lymphoid enhancer factor and then affect the expression of Wnt target genes, including cyclin D1 and c-Myc, eventually promoting tumor pathogenesis [25,26]. β-Catenin is the core member of the Wnt pathway that has been shown to be highly expressed in high-grade glioma and poorly expressed in low-grade astrocytoma, suggesting that β-catenin expression is associated with the degree of malignancy in glioma [27]. There was evidence that the knockdown of β-catenin greatly changed the growth and cell cycle distribution in glioma, inhibiting the proliferation and growth of glioma cells [28]. Moreover, c-Myc is the downstream target gene of the pathway and its enhanced expression was shown to be closely related to the development and progression of glioma [29]. In glioma cells, inhibition of cyclin D1 blocked progression of the cell cycle, inhibited proliferation and induced apoptosis [30,31]. Based on all this evidence, we may conclude that OIP5-AS1 siRNA specifically inhibited the Wnt-7b/β-catenin pathway and the downstream genes cyclin D1 and c-Myc, resulting in cell cycle arrest, thereby inhibiting cell proliferation, invasion and migration, and promoting apoptosis. Finally, similar results were also observed in the nude mice tumorigenesis model, where tumor volume and weight were significantly reduced and the growth was apparently inhibited in mice treated with OIP5-AS1 siRNA, further confirming the inhibitory effect of OIP5-AS1 siRNA on glioma growth.

Therefore, silencing OIP5-AS1 may inhibit the Wnt-7b/β-catenin pathway by up-regulating miR-410, thereby suppressing glioma growth, invasion and migration and promoting apoptosis, deepening understanding of the molecular mechanism of glioma.

Acknowledgements

We would like to express our sincere appreciation to the reviewers for their constructive comments on this paper.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

The authors declare that there are no sources of funding to be acknowledged.

Author Contribution

Wei-Li Sun designed the study, Tian Kang and Yuan-Yu Wang conceived and supervised the study, Jian-Ping Sun and Chen Li performed the examination and the analysis, Hong-Jiang Liu performed the statistical analysis, Yue Yang interpreted the results, and Bao-Hua Jiao drafted the paper. All authors read and approved the final paper.

Abbreviations

     
  • HE

    hematoxylin–eosin

  •  
  • lncRNA

    long noncoding RNA

  •  
  • OIP5-AS1

    OPA-interacting protein 5 antisense transcript 1

References

References
1.
Carro
M.S.
,
Lim
W.K.
,
Alvarez
M.J.
,
Bollo
R.J.
,
Zhao
X.
,
Snyder
E.Y.
et al.
(
2010
)
The transcriptional network for mesenchymal transformation of brain tumours
.
Nature
463
,
318
325
[PubMed]
2.
Beauchesne
P.
(
2012
)
Fotemustine: a third-generation nitrosourea for the treatment of recurrent malignant gliomas
.
Cancers
4
,
77
87
[PubMed]
3.
Stupp
R.
,
Hegi
M.E.
,
Mason
W.P.
,
van den Bent
M.J.
,
Taphoorn
M.J.
,
Janzer
R.C.
et al.
(
2009
)
Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial
.
Lancet Oncol.
10
,
459
466
[PubMed]
4.
Jansen
M.
,
Yip
S.
and
Louis
D.N.
(
2010
)
Molecular pathology in adult gliomas: diagnostic, prognostic, and predictive markers
.
Lancet Neurol.
9
,
717
726
[PubMed]
5.
Hwang
J.H.
,
Smith
C.A.
,
Salhia
B.
and
Rutka
J.T.
(
2008
)
The role of fascin in the migration and invasiveness of malignant glioma cells
.
Neoplasia
10
,
149
159
[PubMed]
6.
Verhaak
R.G.
,
Hoadley
K.A.
,
Purdom
E.
,
Wang
V.
,
Qi
Y.
,
Wilkerson
M.D.
et al.
(
2010
)
Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1
.
Cancer Cell
17
,
98
110
[PubMed]
7.
Argyriou
A.A.
and
Kalofonos
H.P.
(
2009
)
Molecularly targeted therapies for malignant gliomas
.
Mol. Med.
15
,
115
122
[PubMed]
8.
Spizzo
R.
,
Almeida
M.I.
,
Colombatti
A.
and
Calin
G.A.
(
2012
)
Long non-coding RNAs and cancer: a new frontier of translational research?
Oncogene
31
,
4577
4587
9.
Li
J.
,
Zhang
M.
,
An
G.
and
Ma
Q.
(
2016
)
LncRNA TUG1 acts as a tumor suppressor in human glioma by promoting cell apoptosis
.
Exp. Biol. Med.
241
,
644
649
[PubMed]
10.
Zhang
T.
,
Wang
Y.R.
,
Zeng
F.
,
Cao
H.Y.
,
Zhou
H.D.
and
Wang
Y.J.
(
2016
)
LncRNA H19 is overexpressed in glioma tissue, is negatively associated with patient survival, and promotes tumor growth through its derivative miR-675
.
Eur. Rev. Med. Pharmacol. Sci.
20
,
4891
4897
[PubMed]
11.
Naemura
M.
,
Kuroki
M.
,
Tsunoda
T.
,
Arikawa
N.
,
Sawata
Y.
,
Shirasawa
S.
et al.
(
2018
)
The long noncoding RNA OIP5-AS1 is involved in the regulation of cell proliferation
.
Anticancer Res.
38
,
77
81
[PubMed]
12.
Kim
J.
,
Abdelmohsen
K.
,
Yang
X.
,
De
S.
,
Grammatikakis
I.
,
Noh
J.H.
et al.
(
2016
)
LncRNA OIP5-AS1/cyrano sponges RNA-binding protein HuR
.
Nucleic Acids Res.
44
,
2378
2392
[PubMed]
13.
Yang
N.
,
Chen
J.
,
Zhang
H.
,
Wang
X.
,
Yao
H.
,
Peng
Y.
et al.
(
2017
)
LncRNA OIP5-AS1 loss-induced microRNA-410 accumulation regulates cell proliferation and apoptosis by targeting KLF10 via activating PTEN/PI3K/AKT pathway in multiple myeloma
.
Cell Death Dis.
8
,
e2975
14.
Chen
L.
,
Zhang
J.
,
Feng
Y.
,
Li
R.
,
Sun
X.
,
Du
W.
et al.
(
2012
)
MiR-410 regulates MET to influence the proliferation and invasion of glioma
.
Int. J. Biochem. Cell Biol.
44
,
1711
1717
15.
Shiah
S.G.
,
Hsiao
J.R.
,
Chang
W.M.
,
Chen
Y.W.
,
Jin
Y.T.
,
Wong
T.Y.
et al.
(
2014
)
Downregulated miR329 and miR410 promote the proliferation and invasion of oral squamous cell carcinoma by targeting Wnt-7b
.
Cancer Res.
74
,
7560
7572
[PubMed]
16.
Ulitsky
I.
,
Shkumatava
A.
,
Jan
C.H.
,
Sive
H.
and
Bartel
D.P.
(
2011
)
Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution
.
Cell
147
,
1537
1550
[PubMed]
17.
Liu
X.
,
Zheng
J.
,
Xue
Y.
,
Yu
H.
,
Gong
W.
,
Wang
P.
et al.
(
2018
)
PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop regulates the biological behavior of glioma cells
.
Theranostics
8
,
1084
1105
[PubMed]
18.
Bayne
K.
(
1996
)
Revised Guide for the Care and Use of Laboratory Animals available. American Physiological Society.
Physiologist
39
,
199
,
208-111
19.
Louis
D.N.
,
Ohgaki
H.
,
Wiestler
O.D.
,
Cavenee
W.K.
,
Burger
P.C.
,
Jouvet
A.
et al.
(
2007
)
The 2007 WHO classification of tumours of the central nervous system
.
Acta Neuropathol.
114
,
97
109
[PubMed]
20.
Deng
J.
,
Deng
H.
,
Liu
C.
,
Liang
Y.
and
Wang
S.
(
2018
)
Long non-coding RNA OIP5-AS1 functions as an oncogene in lung adenocarcinoma through targeting miR-448/Bcl-2
.
Biomed. Pharmacother.
98
,
102
110
21.
Zhang
H.
,
Qi
Y.
,
Geng
D.
,
Shi
Y.
,
Wang
X.
,
Yu
R.
et al.
(
2018
)
Expression profile and clinical significance of Wnt signaling in human gliomas
.
Oncol. Lett.
15
,
610
617
[PubMed]
22.
Griveau
A.
,
Seano
G.
,
Shelton
S.J.
,
Kupp
R.
,
Jahangiri
A.
,
Obernier
K.
et al.
(
2018
)
A glial signature and Wnt7 signaling regulate glioma-vascular interactions and tumor microenvironment
.
Cancer Cell
33
,
874.e877
889.e877
23.
Arensman
M.D.
,
Kovochich
A.N.
,
Kulikauskas
R.M.
,
Lay
A.R.
,
Yang
P.T.
,
Li
X.
et al.
(
2014
)
WNT7B mediates autocrine Wnt/beta-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma
.
Oncogene
33
,
899
908
[PubMed]
24.
Wang
Z.
,
Shu
W.
,
Lu
M.M.
and
Morrisey
E.E.
(
2005
)
Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5
.
Mol. Cell. Biol.
25
,
5022
5030
[PubMed]
25.
Baarsma
H.A.
,
Konigshoff
M.
and
Gosens
R.
(
2013
)
The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets
.
Pharmacol. Therap.
138
,
66
83
26.
Baryawno
N.
,
Sveinbjornsson
B.
,
Eksborg
S.
,
Chen
C.S.
,
Kogner
P.
and
Johnsen
J.I.
(
2010
)
Small-molecule inhibitors of phosphatidylinositol 3-kinase/Akt signaling inhibit Wnt/beta-catenin pathway cross-talk and suppress medulloblastoma growth
.
Cancer Res.
70
,
266
276
[PubMed]
27.
Liu
C.
,
Tu
Y.
,
Sun
X.
,
Jiang
J.
,
Jin
X.
,
Bo
X.
et al.
(
2011
)
Wnt/beta-Catenin pathway in human glioma: expression pattern and clinical/prognostic correlations
.
Clin. Exp. Med.
11
,
105
112
[PubMed]
28.
Liu
X.
,
Wang
L.
,
Zhao
S.
,
Ji
X.
,
Luo
Y.
and
Ling
F.
(
2011
)
beta-Catenin overexpression in malignant glioma and its role in proliferation and apoptosis in glioblastma cells
.
Med. Oncol.
28
,
608
614
[PubMed]
29.
Zhao
T.
,
Yang
H.
,
Tian
Y.
,
Xie
Q.
,
Lu
Y.
,
Wang
Y.
et al.
(
2016
)
SOX7 is associated with the suppression of human glioma by HMG-box dependent regulation of Wnt/beta-catenin signaling
.
Cancer Lett.
375
,
100
107
[PubMed]
30.
Sun
G.
,
Shi
L.
,
Yan
S.
,
Wan
Z.
,
Jiang
N.
,
Fu
L.
et al.
(
2014
)
MiR-15b targets cyclin D1 to regulate proliferation and apoptosis in glioma cells
.
BioMed Res. Int.
2014
,
687826
[PubMed]
31.
Du
Z.
,
Tong
X.
and
Ye
X.
(
2013
)
Cyclin D1 promotes cell cycle progression through enhancing NDR1/2 kinase activity independent of cyclin-dependent kinase 4
.
J. Biol. Chem.
288
,
26678
26687
[PubMed]
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