The present study was approved by the Ethics Committee of Xiangya Hospital, Central South University (Changsha, China). A total of 58 cases of glioma specimens and 10 cases of normal brain tissues were obtained from Xiangya Hospital, Central South University (Changsha, China) between January 2010 and March 2012. Written informed consent was obtained from all patients prior to the study. Tissues were snap-frozen in liquid nitrogen following surgical resection, and stored in liquid nitrogen until use. The clinicopathological information of the patients with glioma included in the present study is summarized in Table I.

Total RNA was extracted from the tissues and cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The MirVana™ RT-PCR microRNA detection kit (Thermo Fisher Scientific, Inc.) was used to examine miR expression, according to the manufacturer's protocol. U6 was used as an internal reference. The standard SYBR Green RT-PCR Kit (Takara Bio, Inc., Otsu, Japan) was used to examine mRNA expression, according to the manufacturer's protocol. GAPDH was used as an internal reference. The primers used were as follows: miR-423 forward, 5′-ATGGTTCGTGGGTGAGGGGCAGAGAGCGAGAGCAGGGTCCGAGGTATTCG-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; PANX2 forward, 5′-CCAAGAACTTCGCAGAGGAAC-3′ and reverse, 5′-GGGCAGGAACTTGTGCTCA-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The reaction mixture included cDNA solution (1 µl), PCR master mix (10 µl), primers (2 µl) and water (7 µl). The reaction conditions were 9°C for 3 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. The relative expression level was quantified using the 2^−ΔΔCq method (21).

Human glioma U251 and U87MG Uppsala cell lines were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and were maintained at 37°C in a humidified incubator (Thermo Fisher Scientific, Inc.) containing 5% CO₂.

Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) was used to perform transfection, according to the manufacturer's protocol. Briefly, U251 and U87MG Uppsala cells were transfected with a negative control (NC) inhibitor, miR-423-3p inhibitor, non-specific small interfering RNA (siRNA) or PANX2-specific siRNA, respectively were purchased from Guangzhou FulenGen Co. Ltd. (Guangzhou, China). Following transfection at 37°C for 48 h, the expression assay was performed.

U251 and U87MG Uppsala cells were lysed in RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was quantified using a bicinchinonic acid protein assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein (50 µg) was separated by SDS-PAGE (12% gel), transferred onto a polylvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.), and then blocked using 5% non-fat dried milk (Yili Group, Beijing, China) in Tris-buffered saline with Tween-20 (TBST; Beyotime Institute of Biotechnology) at room temperature for 3 h. The membrane was incubated with rabbit polyclonal anti-PANX2 primary antibody (1:100; cat no. ab55917; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-GAPDH primary antibody (1:100; cat no. ab9485; Abcam) at room temperature for 3 h, and then washed three times using TBST. Subsequently, the membrane was incubated with goat monoclonal anti-rabbit secondary antibody (1:5,000; cat no. ab190492; Abcam) for 1 h at room temperature, and washed three times using TBST. The immune complexes were detected using an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Image J software (version 1.0, National Institutes of Health, Bethesda, MD, USA) was used to analyze the relative protein expression, represented as the density ratio relative to GAPDH.

U251 and U87MG Uppsala cells (2×10³ cells per well) were seeded in 96-well plates, and 100 µl fresh serum-free DMEM with 0.5 g/l MTT solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added. Following incubation at 37°C for 0, 24, 48 and 72 h, the medium containing MTT solution was removed, and 50 µl dimethylsulfoxide (Sigma-Aldrich; Merck KGaA) was added. Following incubation at 37°C for 10 min, the absorbance at 570 nm of each sample was determined using a plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A flow cytometer was used to determine the cell apoptosis with an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA). Cells were harvested and washed with ice-cold PBS twice, and 10⁶ cells were resuspended in 200 µl binding buffer with 10 µl annexin-V-FITC and 5 µl propidium iodide-phycoerythrin, prior to incubation in the dark at 4°C for 30 min. Subsequently, 300 µl binding buffer was added, followed by analysis using flow cytometry (BD Accuri C6 software 1.0, C6; BD Biosciences, Franklin Lakes, NJ, USA).

TargetScan Human 5.1 software (22) (www.targetscan.org) was used to determine the putative target of miR-423-3p. The wild-type (WT) PANX2 3′-untranslated region (UTR) was constructed by PCR, which was performed by Yearthbio (Changsha, China), and inserted into the pMIR-REPORT miRNA Expression Reporter vector (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The mutant type (MT) of PANX2 3′-UTR was constructed using the Easy Mutagenesis System kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol, and then inserted into the pMIR-REPORT miRNA Expression Reporter vector (Thermo Fisher Scientific, Inc.). U251 and U87MG Uppsala cells were co-transfected with WT PANX2-3′UTR plasmid or MT PANX2-3′UTR plasmid, and miR-NC or miR-423-3p mimics, using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.). Following transfection at 37°C for 48 h, the activity of Renilla luciferase and firefly luciferase were determined using a dual-luciferase reporter assay system (Promega Corporation), 48 h after transfection. The activity of Renilla luciferase was normalized to that of the firefly luciferase.

Data are expressed as the mean ± standard deviation. The differences between two groups were analyzed using two-tailed Student's t-test. Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). Pearson's correlation analysis was performed to examine the correlation between the miR-423-3p and PANX2 expression in glioma tissues. Kaplan Meier analysis with log rank tests were used for the survival analysis. P<0.05 was considered to indicate a statistically significant difference.

In the present study, qPCR was used to determine the expression of miR-423-3p in glioma tissues. Normal brain tissues were used as controls. As indicated in Fig. 1A, miR-423-3p expression levels were significantly increased in glioma tissues compared with normal brain tissues. Furthermore, the expression of miR-423-3p was increased in World Health Organization (WHO) III–IV grade glioma compared with WHO I–II grade glioma (Fig. 1B). These glioma tissues were further divided into two groups, a high miR-423-3p group and a low miR-423-3p group, on the basis of mean expression value. It was observed that high expression of miR-423-3p was significantly associated with an advanced grade of glioma as well as a low Karnofsky performance score (KPS), but not with age and sex (Table I). Furthermore, as presented in Fig. 1C, the patients with glioma with high miR-423-3p expression levels had a shorter survival time, compared with those with low miR-423-3p expression levels. Therefore, miR-423-3p upregulation may contribute to the malignant progression of glioma as well as poor prognosis in patients with glioma.

As miR-423-3p was upregulated in glioma, glioma U251 and U87MG Uppsala cells were transfected with an miR-423-3p inhibitor in order to knock down miR-423-3p expression. Transfection with an NC inhibitor was used as the control group. Following transfection with miR-423-3p inhibitor, miR-423-3p expression levels were significantly decreased compared with the NC inhibitor group (Fig. 2A and B). An MTT assay further indicated that the inhibition of miR-423-3p led to the decreased proliferation of U251 and U87MG Uppsala cells (Fig. 2C and D). Cell apoptosis was further examined and it was revealed that the knockdown of miR-423-3p resulted in a significant increase in U251 and U87MG Uppsala cell apoptosis (Fig. 2E and F). Therefore, the results of the present study demonstrated that the knockdown of miR-423-3p decreases U251 and U87MG Uppsala cell proliferation and induces cell apoptosis.

Bioinformatics analysis indicated that PANX2 was a putative target gene of miR-423-3p (Fig. 3A). To the best of our knowledge, this targeting association has never previously been reported. In the present study, luciferase reporter plasmids were constructed containing WT or MT PANX2 3′-UTR (Fig. 3B and C). The luciferase reporter gene assay was then performed in U251 and U87MG Uppsala cells. The results of the present study indicated that luciferase activity was significantly decreased in U251 and U87MG Uppsala cells co-transfected with the WT PANX2 3′-UTR plasmid and miR-423-3p mimic compared with the control group, which was eliminated during transfection with the MT PANX2 3′-UTR plasmid (Fig. 3D and E). Subsequently, it was revealed that the inhibition of miR-423-3p significantly increased the expression of PANX2 protein in U251 and U87MG Uppsala cells (Fig. 3F and G). Therefore, PANX2 is a potential novel target gene of miR-423-3p in U251 and U87MG Uppsala cells.

On the basis of the aforementioned results, it was hypothesized that PANX2 may be involved in miR-423-3p-mediated glioma growth. To investigate this hypothesis, U251 and U87MG Uppsala cells were co-transfected with miR-423-3p inhibitor and PANX2 siRNA, or miR-423-3p inhibitor and NC siRNA. Following transfection, the mRNA and protein levels of PANX2 were significantly decreased in the miR-423-3p inhibitor + PANX2 siRNA group, compared with the miR-423-3p inhibitor + NC siRNA group (Fig. 4A-D). An MTT assay further demonstrated that the proliferation of U251 and U87MG Uppsala cells was significantly increased in the miR-423-3p inhibitor + PANX2 siRNA group, compared with the miR-423-3p inhibitor + NC siRNA group (Fig. 5A and B), indicating that knockdown of PANX2 attenuates the suppressive effects of miR-423-3p inhibition on glioma cell proliferation. Cell apoptosis was then assessed. It was revealed that the apoptosis of U251 and U87MG Uppsala cells was significantly decreased in the miR-423-3p inhibitor + PANX2 siRNA group, compared with the miR-423-3p inhibitor + NC siRNA group (Fig. 5C and D), indicating that the knockdown of PANX2 attenuates the effect of miR-423-3p inhibition on glioma cell apoptosis. As a result, it may be suggested that the knockdown of miR-423-3p at least partially inhibits proliferation and induces the apoptosis of glioma cells, via the direct targeting of PANX2.

Finally, RT-qPCR was performed to examine the mRNA expression levels of PANX2 in glioma. The expression of PANX2 mRNA was demonstrated to be significantly decreased in glioma tissues compared with normal brain tissues (Fig. 6A). Furthermore, PANX2 mRNA expression levels in WHO III–IV grade glioma were decreased compared with in WHO I–II grade glioma (Fig. 6B). Notably, the PANX2 mRNA expression levels were inversely correlated with the miR-423-3p expression levels in glioma tissues (Fig. 6C). The decreased expression of PANX2 may be due to the upregulation of miR-423-3p in glioma.