miR‑489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways.

Glioma patients receiving therapy are at a high risk of relapse and rapid progression and, thus, more effective treatments are required. The aim of the present study was to determine the suppressive role of miR‑489 as an alternative therapeutic target for preventing glioma progression. The results of the present study demonstrated that patients with relatively lower levels of expression of miR‑489 had more favorable clinical outcomes. Furthermore, miR‑489 expression was inversely correlated with p21‑activated kinase 5 (PAK5) mRNA expression levels in glioma specimens. A dual luciferase reporter assay revealed that miR‑489 suppressed PAK5 expression by directly targeting the PAK5 3'‑untranslated region. The effects of miR‑489 on cell viability were measured using MTT and Cell Counting Kit‑8 assays. The results demonstrated that ectopic expression of miR‑489 mimic decreased cell viability by interfering with cyclin D1 and c‑Myc signaling. Additionally, the effect of miR‑489 on apoptosis was determined using Hoechst 33258 staining and flow cytometry. The results demonstrated that miR‑489 decreased the activity of RAF1, reduced Bcl‑2 and promoted Bax expression, resulting in increased cell apoptosis. Furthermore, the effect of miR‑489 mimic on cellular motility was assessed using migration and invasion assays. miR‑489 was shown to abolish the PAK5/RAF1/MMP2 pathway, resulting in decreased cell invasion ability. These results indicated that miR‑489 may be involved in PAK5‑mediated regulation of glioma progression, demonstrating the potential therapeutic benefits of targeting miR‑489 in glioma.

Introduction

Glioma is the most common and aggressive malignant primary tumor of the human central nervous system (1,2). Presently available therapies include surgical resection, radiation, chemotherapy and combination therapies. However, despite significant advances in treatment options, patients with glioma frequently display rapid progression and a high rate of recurrence after the initial resection (3). An improved understanding of the underlying molecular pathology and signaling pathways involved in the progression of glioma may uncover novel potential targets in order to design innovative therapies for preventing glioma recurrence and, thus, prolonging survival (4).

p21-activated kinase 5 (PAK5) is a member of the PAK family of Ser/Thr protein kinases. PAK5 is activated by Cdc42/RAC1 and a range of effectors, including hepatocyte growth factor and epidermal growth factor (5). PAK5 is predominantly expressed in the brain and plays a role in multiple signaling pathways, including cytoskeletal regulation, cell motility, cell survival, apoptosis and proliferation (6). PAK5 upregulation is frequently observed in patients with glioma, and has been demonstrated to contribute to glioma cell survival, anti-apoptosis, invasion and progression (7,8). Studies of PAK5 in glioma, particularly on the effects of inactivation of PAK5, are limited.

A wide range of microRNAs (miRNAs) have been demonstrated to be involved in the progression of different types of malignancies (9,10). The aim of the present study was to identify PAK5-targeting miRNAs, and to assess whether these miRNAs possess prognostic or therapeutic value in glioma. miR-489, a PAK5-targeting miRNA, has been shown to regulate glioblastoma progression through the LINC01446/miR-489-3p/TPT1 axis (11). As one of the effectors of the long non-coding RNA (lncRNA) ENST01108, miR-489 targets SIK1 and suppresses glioma progression (12). By targeting SPIN1-mediated regulation of the PI3K/AKT pathway, miR-489 induces apoptosis and arrests cell cycle progression (13). Recent studies have demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic cascades in a number of different types of cancer, indicating that miR-489 targets different oncogenes in glioma as a tumor suppressor miRNA. The aim of the present study was to determine whether PAK5 is a target gene of miR-489, and elucidate the mechanism through which miR-489 suppresses glioma progression.

Materials and methods

Patients

A total of 40 glioma tissues and matched adjacent tissues were used in the present study. The diagnosis of glioma was performed by surgeons and pathologists. The exclusion criteria were as follows: Patients with recurrent glioma; patients who received immunotherapy, radiotherapy or chemotherapy prior to surgery; and other severe organ or autoimmune diseases. The protocol of the present study was approved by The Human Ethics Committee of The First Affiliated Hospital of China Medical University. All the experiments were performed in accordance with the Declaration of Helsinki and subsequent updates. Written informed consent was obtained from each patient.

miRNA microarray analysis

A total of 500 ng of RNA was subjected to Custom RT2 Profiler PCR Array (cat. no. 330171; Qiagen China Co., Ltd.). According to the list of gene names, symbols, UniGene and Genbank IDs, primers were synthesized by the manufacturer (GenePharma Co., Ltd.). Three biological replicates (experimental replicate) were used per group, and each was measured in duplicate (technical replicates). Comparisons for significance were performed using a Student's t-test.

Cell culture and transfection

The normal human astrocyte cell line CC-2565 was obtained from Lonza Group, Ltd. U87 MG cells, a glioblastoma cell line of unknown origin, were obtained from the American Type Culture Collection (cat. no. HTB-14). The human glioma cell line U251 (cat. no. KCB 200965YJ) was purchased from Kunming Institute of Zoology, Chinese Academy of Sciences. Normal human astrocytes (NHA) were cultured in Astrocyte Growth Medium (Lonza Group Ltd.) supplemented with 0.03% FBS (Thermo Fisher Scientific, Inc.). U87 and U251 cells were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Cells were maintained at 37°C in an incubator with a humidified atmosphere with 5% CO2 and 95% air. All the reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.). miR-489 mimics, miR-489 inhibitor (miR-489 inh) and negative control RNA were purchased from GenePharma Co., Ltd. The oligonucleotide sequences were as follows: miR-489 mimic, 5′-GUGACAUCACAUAUACGGCAGC-3′; miR-489 inh, 5′-GCTGCCGUAUAUGUGAUGUCAC-3′; negative control (control), 5′-UUGUCCGAACGUGUCACGUTT-3′. Cells were transfected with Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. pDONR223 PAK5 (Addgene plasmid no. 60532) was a gift from Dr John Brognard (Professor of Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester) (14). pDONR223 (Addgene plasmid no. 12536017) was obtained from Thermo Fisher Scientific, Inc.

Luciferase reporter assays

Wild-type (wt) or mutant (mut) 3′-untranslated region (3′-UTR) of the PAK5 promoter was amplified and cloned into a pGL474-vector, and the 3′-UTR of PAK5 and corresponding miRNA vectors were co-transfected into U87 and U251 cells, respectively. The primer sequences were as follows: Wt-PAK5 forward, 5′-CTTTGATGTCATGTAGCCATTG-3′ and reverse, 5′-CAAAGTTCCTAAAGAATCATTGGT-3′; and mut-PAK5 forward, 5′-CTTTGATGTCCTAATGCCATTG-3′ and reverse, 5′-CAAAGTTCCTAATTAGCATTGGT-3′. At 24 h after co-transfection, luciferase activity was measured using a Dual-Luciferase® Reporter Assay System (Promega Corporation).

Reverse transcription-quantitative (RT-q) PCR analysis

Total RNA was extracted using TRIzol® (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription of RNA to cDNA was performed using a reverse transcription system (Promega Corporation). PCR amplification of miR-489 and PAK5 mRNA was performed using an Mx3000P real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. U6 and GAPDH were used as the reference genes to normalize the expression of miR-489 and PAK5 mRNA, respectively. The primer sequences for the detection of mRNA expression were as follows: PAK5: Forward, 5′-GTCTCCTCTGACTTCAACAGC-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′; miR-489: Forward, 5′-GTGACATCACATATACGG-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′; U6: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; c-Myc: Forward, 5′-CCTGGTGCTCCATGAGGAGAC-3′ and reverse, 5′-CAGACTCTGACCTTTTGCCAGG-3′; cyclin D1: Forward, 5′-ATGTTCGTGGCCTCTAAGATG-3′ and reverse, 5′-CAGGTTCCACTTGAGCTTGTTC-3′; Bcl-2: Forward, 5′-ATCGCCCTGTGGATGACTGAG-3′ and reverse, 5′-CCAGGAGAAATCAAACAGAGG-3′; Bax: Forward, 5′-TCAGGATGCGTCCACCAAGAA-3′ and reverse, 5′-TGTGTCCACGGCGGCAATCATC-3′; MMP2: Forward, 5′-AGCGAGTGGATGCCGCCTTTA-3′ and reverse, 5′-CATTCCAGGCATCTGCGATGAG-3′; GAPDH: Forward, 5′-GTCTCCTCTGACTTCAACAGC-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

Western blotting

Total proteins from 1×106 cells were extracted using lysis buffer (Thermo Fisher Scientific, Inc.) and protein concentration was determined using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). A total of 40 µg protein was resolved on a 10% gel using SDS-PAGE (Thermo Fisher Scientific, Inc.). Subsequently, proteins were transferred to a PVDF membrane (EMD Millipore). The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam). GAPDH was used as the loading control (mouse monoclonal anti-GAPDH, KC-5G4, obtained from Zhejiang Kangchen Biotech Co., Ltd.). The dilution of primary antibodies was 1:1,000 and the dilution of secondary antibodies was 1:5,000. Signals were visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.).

MTT assay

Glioma cell lines were transfected with negative control (NC), miR-489 mimic or miR-489 inhibitor using Lipofectamine® 3000. After 24 h, 1×104 cells were plated in medium containing 10% FBS in 96-well plates. Cell proliferation was measured using a modified MTT assay. Briefly, MTT solution in PBS was added to each well. After a 4 h incubation at 37°C, the medium was replaced with DMSO. After a 15 min incubation at 37°C, the optical density at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell Counting Kit-8 (CCK-8) assay

Glioma cells transfected with miR-489 mimic or inh were plated at a density of 2×103 cells/well into 96-well plates at 37°C in an incubator with 5% CO2. Cell proliferation was assessed every 24 h using a CCK-8 assay (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. For each sample at each time-point, six wells were analyzed and the experiment was repeated three times.

Migration and invasion assays

Migration and invasion assays were performed using modified Boyden chambers with Corning® Transwell® polycarbonate membrane cell culture inserts (Sigma-Aldrich; Merck KGaA). The invasion assay was performed using BioCoat Matrigel invasion chambers (BD Biosciences). A total of 1×105 cells in 100 µl serum-free DMEM supplemented with 0.1% BSA were placed in the upper part of each chamber, and the lower compartments were filled with 600 µl DMEM supplemented with 10% FBS. The cells were incubated for 18 h at 37°C, and the cells that had invaded or migrated through the membrane were stained with 0.5% crystal violet solution and counted.

Hoechst 33258 staining

Glioma cells were transfected with NC, miR-489 mimic, or miR-489 inh for 24 h. After incubation, the cells were fixed with 4% polyoxymethylene, washed twice with PBS, treated with 10 µg/ml Hoechst 33258 (Molecular Probes; Thermo Fisher Scientific, Inc.) for 5 min at room temperature, and subsequently washed with PBS three times, for 10 min per wash. Cells were observed using a fluorescence microscope (Leica Microsystems GmbH).

Statistical analysis

The results are presented as the mean ± standard deviation of at least three experimental repeats. Comparisons among different groups were performed using a two-way ANOVA. Dunnett's multiple comparisons test was performed to compare the mean of each group with the mean of the control group. Paired data were compared using a Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Association between miR-489 and PAK5 in glioma

Recently, numerous miRNAs have been demonstrated to be aberrantly expressed in glioma tissues compared with adjacent tissues. In the present study, it was demonstrated that the difference in miR-489 expression between glioma and adjacent tissues was the largest amongst the various miRNAs measured (Table I). Using the miRDB database (9,15), miR-489 was identified as a candidate miRNA targeting PAK5. The relative RNA expression levels of miR-489 and PAK5 were determined in 40 samples from patients with glioma (Fig. 1). The results demonstrated that the expression of miR-489 was decreased in cancer tissues (0.583±0.080) compared with that in adjacent tissues (0.774±0.110), whereas the expression of PAK5 increased in cancer tissues (0.732±0.072) compared with that in adjacent tissues (0.572±0.070) (Fig. 1A and C). To determine whether there was an association between miR-489 expression and clinical outcome in patients with glioma, the 40 cases were divided into two groups according to miR-489 expression, the high miR-489 (n=14) and low miR-489 (n=26) groups. As shown in Fig. 1B, patients with higher miR-489 expression levels had longer survival. In addition, as shown in Fig. 1D, patients with lower PAK5 expression levels (n=20) had longer survival.

Table I.

Differentially expressed miRNAs in glioma tissues vs. adjacent tissues.

Name	Fold change	Up/downregulation	P-value
miR-489-3p	−48.6678	Down	0.007856
miR-148b-5p	−29.7516	Down	0.001545
miR-505-3p	−22.5996	Down	0.000132
miR-182-3p	−18.5699	Down	0.02908
miR-21-5p	−9.01	Down	0.010861
miR-125a-3p	−7.6117	Down	0.000436
miR-93-5p	27.3139	Up	0.00837
miR-140-3p	16.7361	Up	0.002108
miR-148a-3p	9.8826	Up	0.01715

P<0.05 was considered statistically significant. Data were obtained from three independent experiments.

Figure 1.

miR-489 expression is negatively correlated with PAK5 expression and predicts a more favorable clinical outcome in patients with glioma. (A) Relative RNA expression levels of miR-489 in 40 glioma and adjacent matched healthy tissues. miR-489 expression was lower in glioma tissues compared with the matched adjacent tissues. **P<0.01 vs. adjacent tissues. (B) Kaplan-Meier survival analysis demonstrated that upregulated miR-489 expression levels were associated with longer overall survival in patients with glioma. (C) mRNA expression levels of PAK5 in the 40 glioma specimens and adjacent tissues. PAK5 expression was higher in the glioma tissues compared with that in matched adjacent tissues. **P<0.01 vs. adjacent tissues. (D) Kaplan-Meier survival analysis revealed that upregulated expression of PAK5 was associated with shorter overall survival in patients with glioma. (E) Association between relative RNA expression levels of miR-489 and PAK5 in 40 patients. PAK5, p21-activated kinase 5.

To further determine the association between miR-489 and PAK5 in glioma, a Spearman's rank correlation analysis was performed (Fig. 1E). miR-489 and PAK5 expression were found to be negatively correlated. These results suggest that miR-489 expression is negatively correlated with PAK5 expression and may be used to predict the clinical outcome in patients with glioma.

miR-489 decreases PAK5 expression by targeting the 3′-UTR of PAK5

miRNA target prediction data were analyzed using the miRDB database (http://mirdb.org/index.html), and it was predicted that miR-489 targeted the 3′-UTR of PAK5 (Fig. 2A). A dual luciferase reporter assay was performed in U87 and U251 cells to demonstrate the putative binding sites. Glioma cells were transfected with miR-489 mimic or miR-489 inh, combined with wt-PAK5 promoter or mut-PAK5 promoter.

Figure 2.

miR-489 targets the PAK5 3′-UTR and inhibits the expression of PAK5 in glioma cells. (A) Schematic illustration of the PAK5 3′-UTR with putative binding sites for miR-489. The activity of wt-PAK5 decreased significantly compared with that of the mut-PAK5 promoter in (B) U87 and (C) U251 cells transfected with miR-489 mimic. Quantitative analysis of the luciferase reporter assays is presented as the mean of three independent experiments, each performed in triplicate. Control, non-targeting control short hairpin RNA. **P<0.01 vs. control. Relative expression of (D) miR-489 and (E) PAK5 in NHA, U87 and U251 cells. Data are presented as the mean ± standard deviation. **P<0.01 vs. NHA. (F) U87 and U251 cells were transfected with control, miR-489 mimic or miR-489 inh for 24 h, and the expression of PAK5 was determined by western blotting. GAPDH was used as the loading control. **P<0.01 vs. NHA. (G) U87 cells and (H) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for 24 h, and the relative mRNA expression levels of PAK5 were determined by reverse transcription-quantitative PCR. **P<0.01 vs. control. PAK5, p21-activated kinase 5; wt, wild-type; mut, mutant; NHA, normal human astrocytes; miR-489 inh, mir-489 inhibitor; miR-489, miR-489 mimic; con, control; UTR, untranslated region.

As shown in Fig. 2B, the relative activity of the wt-PAK5 promoter was reduced in cells transfected with miR-489 mimic in U87 cells. Ectopic expression of miR-489 inh restored the relative activity of wt-PAK5 compared with the control. The relative activity of the mut-PAK5 promoter was not altered in cells transfected with either miR-489 mimic or miR-489 inh. Similar results were observed in the U251 cells (Fig. 2C).

To further investigate expression of miR-489 and PAK5 in glioma cells, RT-qPCR assays were performed. As shown in Fig. 2D, miR-489 expression was lower in U87 and U251 cells compared with NHA, whereas PAK5 expression was higher in U87 and U251 cells compared with NHA (Fig. 2E). Additionally, western blotting was performed to examine changes in PAK5 protein expression following transfection with miR-489 mimic or miR-489 inh. PAK5 expression increased following transfection of miR-489 inh, and decreased significantly following transfection with miR-489 mimic in both U87 and U251 cells (Fig. 2F). Similar changes were observed in the relative RNA expression levels (Fig. 2G and H). These results suggest that miR-489 targets the 3′-UTR of PAK5 and decreases the expression of PAK5 in glioma cells.

miR-489 suppresses cell viability through decreasing the expression of PAK5

Recent studies have demonstrated that miR-489 is a tumor suppressor targeting multiple oncogenes in a number of different types of cancer. miR-489 mimic or miR-489 inh was transfected into U87 and U251 cells to determine the effects of miR-489 on glioma cells in vitro. MTT assays were performed to determine the effects of miR-489 on cell viability. The results demonstrated that miR-489 mimic decreased cell viability, whereas miR-489 inh increased the viability of both U87 and U251 cells (Fig. 3A and B). The differences in cell viability after 36 and 48 h were significant. A CCK-8 assay was used to evaluate the effect of miR-489 on cell proliferation. As shown in Fig. 3C, proliferation was increased in cells transfected with miR-489 mimic, whereas it was decreased in cells transfected with miR-489 inh. Similar results were observed in both U251 and U87 cells (Fig. 3D).

Figure 3.

miR-489 inhibits cell viability by attenuating the PAK5-RAF1-MAPK signaling cascade. (A) U87 and (B) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for the indicated times. MTT assays were performed. **P<0.01 vs. control. Cell Counting Kit-8 assay was used to examine the proliferation of (C) U87 and (D) U251 cells transfected with miR-489 mimic or miR-489 inh for the indicated times. **P<0.01 vs. control. (E) Western blotting was used to determine the expression of the indicated proteins in (E) U87 and (F) U251 cells following transfection with control, miR-489 mimic or miR-489 inh for 24 h. (G) RT-qPCR was used to determine the mRNA expression levels of the indicated genes in U87 cells following transfection with control or miR-489 mimic for 24 h. (H) RT-qPCR was used to determine expression of the indicated genes in U87 cells transfected with control or miR-489 inh for 24 h. **P<0.01 vs. control. The results are presented as the mean ± standard deviation of three independent experiments. PAK5, p21-activated kinase 5; miR-489 inh, miR-489 inhibitor; RT-qPCR, reverse transcription-quantitative PCR; con, control.

As PAK5 phosphorylates RAF1, but not B-raf, at Ser338, thereby activating the RAF1/ERK/MAPK signaling pathway (16), the expression of phospho-Ser338 RAF1 (p-RAF1), RAF1, and the effectors c-Myc and cyclin D1 was determined in U87 and U251 cells using western blotting. As shown in Fig. 3E, following Ser338 phosphorylation, c-Myc and cyclin D1 decreased in U87 cells, whereas PAK5 expression was decreased following transfection with miR-489 mimic, although total RAF1 remained unchanged. By contrast, p-RAF1, c-Myc and cyclin D1 expression decreased following transfection with miR-489 inh. Similar results were observed in U251 cells (Fig. 3F). To confirm the changes in the protein expression levels, RT-qPCR assays were performed to evaluate changes in the relative RNA levels. As shown in Fig. 3G, transfection of miR-489 mimic decreased PAK5, c-Myc and cyclin D1 RNA expression levels significantly, but did not affect RAF1 levels in U87 cells. Transfection with miR-489 inh significantly increased PAK5, c-Myc and cyclin D1 levels (Fig. 3H). Similar results were observed in the U251 cells (data not shown). These results suggest that miR-489 decreases cell viability by inactivating PAK5/RAF1-mediated pathways.

miR-489 increases apoptosis by suppressing the RAF1/Bax pathways

As previously described, the pro-oncogene RAF1 and apoptosis-associated proteins Bax/Bcl-2 were involved in the regulation of apoptosis in a variety of different types of cancer (17,18). U87 and U251 cells were transfected with control, miR-489 mimic or miR-489 inh. Hoechst 33258 staining was performed to determine whether miR-489 increased the apoptosis of glioma cells. Increased condensation of chromatin was observed in glioma cells following transfection with the miR-489 mimic (Fig. 4A and C), whereas the opposite was observed in cells transfected with miR-489 inh (Fig. 4B and D). Furthermore, apoptosis was determined by flow cytometry 24 h after transfection, and the results demonstrated that apoptosis in cells transfected with miR-489 mimic was significantly increased, whereas apoptosis in cells transfected with miR-489 inh was significantly decreased compared with control.

Figure 4.

miR-489 promotes apoptosis in glioma cells by attenuating RAF1-Bax-mediated cell survival pathways. (A and B) U87 and (C and D) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for 24 h, and subsequently stained with Hoechst 33258 and observed under a fluorescence microscope. Flow cytometry analysis was performed to measure the apoptosis in transfected cells after 24 h. (E) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h post-transfection. U87 cells were transfected with control, (F) miR-489 mimic or (G) miR-489 inh and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. (H) U251 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (I) miR-489 mimic or (J) miR-489 inh, and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. **P<0.01 vs. control. Data are presented as the mean ± standard deviation of three independent experiments. A two-way ANOVA was used to analyze the differences between two groups. miR-489 inh, miR-489 inhibitor; miR-489, miR-489 mimic; con, control.

Western blotting was performed to assess changes in expression of the pro-apoptotic protein Bax, and pro-survival protein Bcl-2. As shown in Fig. 4E and H, Bax expression decreased significantly, whereas Bcl-2 expression increased significantly following transfection with miR-489 mimic in both U87 and U251 cells. Transfection of miR-489 inh significantly upregulated Bax expression and downregulated Bcl-2 expression. Furthermore, RT-qPCR analysis demonstrated that transfection of miR-489 mimic increased Bax expression and decreased Bcl-2 expression (Fig. 4F), whereas the opposite results were observed in cells transfected with miR-489 inh in U87 cells (Fig. 4G). The effects of miR-489 on the expression of Bax and Bcl-2 in U251 cells were consistent with those in U87 cells (Fig. 4I and J). These results suggest that miR-489 induced apoptosis in glioma cells by inhibiting the PAK5/RAF1/Bax/Bcl-2 axis.

miR-489 suppresses cell motility by decreasing MMP2 expression

To investigate the effects of miR-489 on migration and invasion, Transwell assays with or without Matrigel® were performed. As shown in Fig. 5A and B, miR-489 mimic significantly decreased migration and invasion, whereas miR-489 inh significantly increased migration and invasion in both U87 and U251 cells. The cell counts of invading or migrating cells are presented in Table II. To further verify the results, western blotting and RT-qPCR analyses were performed in U87 and U251 cells. As shown in Fig. 5C-E, MMP2 expression increased significantly following transfection with miR-489 mimic, whereas the opposite was observed in cells transfected with miR-489 inh. Consistently with the results of RNA expression, the protein levels of MMP2 were decreased in cells transfected with miR-489 mimic, whereas they were significantly increased in cells transfected with miR-489 inh. Similar results were observed in U251 cells (Fig. 5F-H).

Figure 5.

miR-489 decreases migration and invasion by attenuating the PAK5-RAF1-MMP2 axis. Migration and invasion assays were performed with (A) U87 and (B) U251 transfected with either control, miR-489 mimic or miR-489 inh. Representative images of migration and invasion are shown. Magnification ×100. (C) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (D) miR-489 mimic or (E) miR-489 inh and the mRNA expression levels of MMP2 were measured at 24 h after transfection. **P<0.05 vs. control. (F) U251 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was performed to determine the expression of the indicated proteins at 24 h after transfection. U251 cells were transfected with control, (G) miR-489 mimic or (H) miR-489 inhibitor and the mRNA expression levels of MMP2 were measured at 24 h after transfection. **P<0.05 vs. control. Data are presented as the mean ± standard deviation of three independent experiments. A two-way ANOVA was used to analyze the differences between two groups. PAK5, p21-activated kinase 5; miR-489 inh, miR-489 inhibitor; MMP2, matrix metalloproteinase 2; miR-489, miR-489 mimic; con, control.

Table II.

Effect of miR-489 on migration and invasion of glioma cells.

A, Effect of miR-489 mimic on glioma cells
	U87			U251
Activity	Control	miR-489 mimic	P-value	Control	miR-489 mimic	P-value
Migration	249±6	213±4	<0.001	215±3	197±5	<0.01
Invasion	192±5	167±5	<0.001	177±6	151±4	<0.001
B, Effect of miR-489 inhibitor on glioma cells
	U87			U251
Activity	Control	miR-489 inhibitor	P-value	Control	miR-489 inhibitor	P-value
Migration	201±3	239±4	<0.01	205±6	231±6	<0.001
Invasion	189±3	221±5	<0.001	177±5	218±3	<0.001

Values are presented as mean ± standard deviation vs. control group. P<0.05 was considered statistically significant. Data were obtained from three independent experiments.

These results suggest that miR-489 decreased cell migration and invasion by attenuating the PAK5-MMP2 signaling pathway.

miR-489 suppresses glioma progression by targeting PAK5 and inhibiting PAK5/RAF1-mediated signaling

U251 cells were transfected with control, miR-489 mimic or a combination of miR-489 mimic and PAK5 for 24 h. MTT assays, flow cytometry and Transwell invasion assays were performed to assess cell viability, apoptosis and invasiveness, respectively. The results demonstrated that overexpression of PAK5 reversed the effects of miR-489 on cell growth (Fig. 6A), apoptosis (Fig. 6B) and invasion (Fig. 6C). Furthermore, western blot analysis demonstrated that the overexpression of PAK5 attenuated the miR-489-induced dephosphorylation of RAF1 (Fig. 6D). Additionally, RT-qPCR analysis confirmed that overexpression of miR-489 decreased the expression of c-Myc, cyclin D1, Bcl-2 and MMP2, whereas miR-489 enhanced Bax expression, which was accompanied by a reduction in PAK5 expression (Fig. 6E). Overexpression of PAK5 enhanced the expression of c-Myc, cyclin D1, Bcl-2 and MMP2, and reduced the expression of Bax. These results suggest that miR-489 targeted PAK5 and downregulated PAK5/RAF1-mediated signaling.

Figure 6.

miR-489 suppresses the progression of glioma by targeting PAK5 and inhibiting PAK5/RAF1-mediated signaling pathways. U251 cells were treated with control, miR-489 mimic or a combination of miR-489 mimic and PAK5. (A) MTT assays were performed and the results are presented as the mean ± standard deviation of three individual experiments. **P<0.01. (B) Flow cytometry analysis was performed on cells at 24 h after transfection. Representative images are shown and all experiments were repeated three times. (C) Invasion assays were performed and representative images were captured. Magnification ×100. The histogram shows the mean ± standard deviation of three independent experiments. **P<0.01, vs. control. ##P<0.01, vs. miR-489. (D) Changes in the expression of PAK5 and the levels of p-RAF1 and total RAF1 were examined by western blotting. (E) Expression of the indicated genes in U251 cells transfected with miR-489 or a combination of miR-489 and PAK5. All experiments were repeated three times. A two-way ANOVA was used to analyze the differences between two groups. **P<0.05 vs. control. PAK5, p21-activated kinase 5; MMP2, matrix metalloproteinase 2; miR-489 inh, miR-489 inhibitor; miR-489, miR-489 mimic; con, control.

Discussion

Despite advances in our understanding of tumorigenesis and progression of glioma, presently available therapeutic approaches cannot cure glioma, and thus contribute little to survival time. At present, molecules and signaling pathways have been identified to be involved in the malignant properties of glioma, such as anti-apoptosis, invasion and chemo-resistance, the critical factors remain to be determined (19–21).

In the present study, miR-489 showed the largest fold change in expression in a miRNA microarray analysis. A number of studies have demonstrated that miR-489 is aberrantly expressed in several types of cancers, including breast cancer (22–26), colorectal cancer (27,28), glioma (11–13), hepatocellular carcinoma (29), melanoma (30), ovarian cancer (31,32) and prostate cancer (33). In particular, Soni et al (22) reported that miR-489 expression levels are associated with poor overall survival in patients with a mutant lysosomal protein transmembrane 4 beta in breast cancer (22). Gao et al (27,28) showed that patients with elevated miR-489 expression levels had reduced cancer free recurrence times.

In the present study, miR-489 expression levels in glioma tissues and matching adjacent normal tissues were determined. miR-489 expression was downregulated in glioma tissues compared with the matching normal tissues. Upregulated levels of miR-489 predicted longer overall survival of patients with glioma. Li et al (13) reported that patients with decreased levels of miR-489 expression had a markedly reduced overall survival (13).

Among 232 predicted targets of miR-489 in miRDB, PAK5, which is expressed predominantly in the central nervous system and regulates multiple cell behaviors, including cytoskeletal stabilization, cell migration, proliferation and cell survival was identified as the candidate (5,34,35). PAK5 expression was determined in the same paired tissues. Compared with matched tumor-adjacent tissues, PAK5 expression was upregulated in cancer tissues significantly, and patients with increased PAK5 expression levels exhibited less favorable clinical outcomes. Consistent with this result, previous studies showed that upregulated PAK5 expression was associated with significantly worse survival in patients with breast cancer (36), bladder cancer (37) and gastric cancer (38). The results of the present study provide evidence that increased PAK5 expression is associated with shorter overall survival in patients with glioma. miR-489 expression was negatively correlated with PAK5 expression. The correlation between miR-489 and PAK5 suggested that miR-489 targeted PAK5 and regulated PAK5-mediated signaling in glioma. There are numerous studies on PAK5 in different types of cancer, although the data of PAK5 in glioma is limited. Increased PAK5 expression in glioma tissues and cells promoted glioma progression by impairing cell cycle arrest and enhancing invasion (7,8). In addition, Zheng et al (39) showed that lncRNA colorectal neoplasia differentially expressed rescued apoptotic suppressor protein XIAP and PAK5 expression by inhibiting miR-186 expression, and thus promoted proliferation, migration, invasion and survival of glioma stem cells. In the present study, it was demonstrated that miR-489 targeted the PAK5 3′-UTR directly using a mut-PAK5 3′-UTR, resulting in suppression of PAK5 expression. Additionally, overexpression of miR-489 attenuated the PAK5/RAF1 axis, resulting in a decrease in cell survival. Overexpression of PAK5 reversed the miR-489 mediated effects on cell growth and invasion, suggesting that regulation of miR-489 on glioma cell growth and invasion is dependent on PAK5. Further experiments with glioma xenografts and integrated analysis of The Cancer Genome Atlas data are required to investigate this hypothesis.

In conclusion, the present study demonstrated that miR-489 was downregulated while PAK5 was upregulated in glioma tissues. miR-489 reduced cell viability and invasion while inducing apoptosis by targeting PAK5/RAF1-mediated pathways. The mechanism underlying the inhibition of malignant behavior was dependent on downregulation of PAK5, improving our understanding of PAK5-mediated signaling cascades in glioma. The present study highlights potentially novel therapeutic targets for treating patients with glioma.