Between March 2016 and April 2017, 20 patients (aged 27.3±9.6, male:female=12:8) with glioma were admitted to Tianjin Medical University General Hospital (Tianjin, China). Fresh high- and low-grade glioma samples (6 cases were high-grade and 14 were low-grade) and adjacent normal tissues were obtained from these patients during a temporal lobectomy procedure for epilepsy and immediately snap-frozen in liquid nitrogen upon surgical removal. The present study was approved by the ethics committee of Tianjin Medical University General Hospital according to the criteria of the Declaration of Helsinki. Informed consent was acquired from each patient.

The human glioma cell line U251 was purchased from the European Collection of Cell Cultures (09063001; ECACC, Porton Down, Salisbury, UK; deposited by Shanghaisixin Biotech Co., Ltd, Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 µg/ml streptomycin and 100 U/ml penicillin in a 5% CO₂ incubator at 37°C.

U251 cells of an appropriate concentration (approximately 80%) at 1×10⁵ were seeded on 6-well plates and were incubated at 37°C in the plates for a further 24 h prior to transfection. Cells were subsequently transfected with appropriate concentrations (200 µM for each) of pcDNA3.1-FTH1P3 (pc-FTH1P3) (Sangon Biotech Co., Ltd., Shanghai, China), small interfering RNA (siRNA)-FTH1P3, miR-224-5p mimic, pcDNA3.1-TPD52 (pc-TPD52) and the corresponding controls using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Medium was replaced following 4 h of transfection. The sequences for the target vectors were: FTH1P3 siRNA, 5′-CACCGCCAGCCCTCCGTCACCTCTTCGAAAAGAGGTGACGGAGGGCTGGC-3′; miR-224-5p mimic sense, 5′-CAAGUCACUAGUGGUUCCGUU-3′ and antisense, 5′-CGGAACCACUAGUGACUUGUU-3′; siRNAcontrol sense, 5′-CACCGGGAGAATGCGATGGGAGAGCCGAAGCTCTCCCATCGCATTCTCCC-3′ and antisense: 5′-AAAAGGGAGAATGCGATGGGAGAGCTTCGGCTCTCCCATCGCATTCTCCC-3′; miRNA mimic control, sense 5′-AAAAUGGUGGUGCCCUAGUGACUACA-3′ and antisense, 5′-UAGUCACCAUAAUAGGGCACCAUUUUUU-3′. Cells were harvested for further experimentation at 24, 48 and 72 h following incubation.

Cell viability was detected with a MTT assay. Transfected cells were seeded in a 96-well plate at a density of 2×10⁴ cells/well at 37°C and allowed to attach overnight. MTT solution (20 µl; 0.5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was subsequently added to each well for 4 h at 37°C. Medium was removed and 0.2 ml dimethyl sulfoxide was added to each well for 30 min at 37°C. The optical density of each well at 490 nm was read on an enzyme-labeled instrument (microplate reader, Bio-Rad Laboratories, Inc., Hercules, CA, USA) to assess cell viability.

Following transfection, cells at a density of 1×10⁵ cells/well were incubated with 10 µM BrdU (Sigma-Aldrich; Merck KGaA) at 37°C for 48 h and fixed in 4% paraformaldehyde at 37°C for 30 min. Cells were subsequently incubated with 0.1% Triton X-100 for 10 min, 2 M HCl for 5 min and 0.1 M borate buffer (pH 8.5) for 5 min in the proper order, as listed, at 37°C. To detect the BrdU-positive cells, cells were blocked with 2% bovine serum albumin (FBS; Gibco; Thermo Fisher Scientific, Inc.), probed with an anti-BrdU antibody (1:1,000; ab1893; Abcam, Cambridge, UK) overnight at 4°C, followed by incubation with a fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin G secondary antibody (1:1,000; ab157532; Abcam). The percentage of cells with positive staining was examined in three randomly selected high-power fields at ×400 magnification using a fluorescence microscope and Cellquest software (version 3.0; BD Biosciences, Franklin Lakes, NJ USA).

The apoptosis rate of each group was examined with an Annexin V-FITC apoptosis detection kit (Beijing Biosea Biotechnology Co., Ltd, Beijing, China), according to the manufacturer's protocol. Briefly, cells (5×10⁶) were harvested, seeded in a 6-well plate and mixed with 10 µl Annexin and 5 µl propidium iodide (10 mg/l) for 15 min in the dark. Apoptosis was detected with a flow cytometer (BD Biosciences) and the apoptotic percentages were analyzed with BD CellQuest 3.0 software (BD Biosciences). Annexin V-positive cells were regarded as apoptotic cells.

TargetScanHuman version 7.1 (http://www.targetscan.org/vert_72/) was used to predict the target sequences of miR-224-5p in TPD52. To construct the wild-type (WT) luciferase reporter vectors, the TPD52 3′-untranslated region (3′-UTR), which included the miR-224-5p seed binding sites, was cloned into the pGL3-promotor vector (Sangon Biotech Co., Ltd., Shanghai, China). In the mutation-type (MUT) luciferase reporter vector, the predicted seed zones in miR-224-5p were replaced by nonsense sequences. For the luciferase reporter assay, 100 ng TPD52 3′UTR luciferase construct and 400 ng miR-224-5p mimics were co-transfected into U251 cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The relative luciferase activity of the luciferase construct was subsequently detected using a Dual-Glo Luciferase assay kit (E2940; Promega Corporation, Madison, WI, USA) following 48 h transfection. Firefly luciferase activity was normalized to Renilla activity. The β-galactosidase expression vector was used as a control for transfection efficiency.

Total RNA was extracted from tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized at 70°C using a Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol [after heating to 94°C for 2 min, the volume (50 µl) was subjected to 30 cycles of 94°C for 30 sec, 61°C for 30 sec, and 72°C for 30 sec]. The gene expression levels of FTH1P3, miR-224-5p and TPD52 were determined by SYBR green qPCR kit (Thermo Fisher Scientific, Inc.) and calculated using the 2^−ΔΔCq method (19). The primer sequences used were as follows: FTH1P3 forward, 5′-TCCATTTACCTGTGCGTGGC-3′ and reverse, 5′-GAAGGAAGATTCGGCCACCT-3′; miR-224-5p forward, 5′-CTGGTAGGTAAGTCACTA-3′ and reverse, 5′-TCAACTGGTGTCGTGGAG-3′; U6 forward, 5′-CTGGTAGGGTGCTCGCTTCGGCAG-3′ and reverse, 5′-CAACTGGTGTCGTGGAGTCGGC-3′; TPD52 forward, 5′-CGTTGTATTTGTTATTTATCAAGTTGT-3′ and reverse, 5′-TCCCCAAGTAAATCTAGTCATGC3′; GAPDH forward, 5′-AAGACCTTGGGCTGGGACTG-3′ and reverse, 5′-ACCAAATCCGTTGACTCCGA-3′. PCR product was produced under the following parameters: 1 Predenaturation cycle of 2 min at 94°C, 32 cycles of 95°C for 15 sec, 64°C for 30 sec, and 72°C for 2 min, with a final extension at 72°C for 5 min. GAPDH was used as the endogenous control for FTH1P3 and TPD52, whereas U6 was used as an internal control for miR-224-5p expression.

Total protein was extracted from cells with radioimmunoprecipitation assay lysis buffer (Sangon Biotech Co., Ltd., Shanghai, China) and the concentration was measured with a bicinchoninic acid protein assay kit. Equal amounts of protein (50 µg per lane) were subjected to 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline with Tween-20 containing 5% non-fat milk at 4°C overnight, and subsequently incubated with primary rabbit monoclonal antibodies against apoptosis regulator Bcl-2 (BCL2; 1:1,000; cat. no. ab32124), apoptosis regulator BAX (BAX; 1:1,000; cat. no. ab32503), TPD52 (1:1,000; cat. no. ab181260) and GAPDH (1:1,000; cat. no. ab8245; Abcam, Cambridge, UK) overnight at 4°C. Following this, the membrane was probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab97051; Abcam) for 2 h at room temperature. An enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to visualize the protein bands. Western blot quantification was performed using ImageJ v1.48 software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as the reference protein.

All experiments were repeated at least three times and the obtained data are presented as the mean ± standard deviation. Student's t-tests were used for the analysis of statistical significance between two groups, and one-way analysis of variance followed by Dunnett's post hoc test was applied to analyze statistical significance among three groups or more. All statistical analysis was conducted using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The expression of FTH1P3 in glioma tissues was initially determined. As presented in Fig. 1A, FTH1P3 expression in glioma tissues was significantly increased compared with that in normal tissues (P<0.001); furthermore, TH1P3 expression in high-grade glioma tissues was significantly higher compared with low-grade glioma tissues (P<0.05), indicating that the upregulation of FTH1P3 may be associated with the development and progression of glioma. Thus, FTH1P3 was overexpressed and suppressed in glioma U251 cells to investigate the potential role of FTH1P3 in glioma in vitro. Following transfection with pc-FTH1P3 and siRNA-FTH1P3, FTH1P3 expression was successfully overexpressed and suppressed, compared with transfection with the corresponding control (P<0.01, FTH1P3 vs. blank control; P<0.001 si-FTH1P3 vs. si-control; Fig. 1B). The results of the MTT assay demonstrated that overexpression of FTH1P3 markedly increased cell viability after 24, 48 and 72 h, whereas suppression of FTH1P3 had the opposite effect (P<0.05 at 24 h, P<0.01 at 48 h, and P<0.001 at 72 h; Fig. 1C). Similar results were obtained from BrdU assay; BrdU-positive cells were significantly increased in the pc-FTH1P3 group compared with the blank group, whereas positive cells were markedly decreased in the siRNA-FTH1P3 group compared with the siRNA control group (P<0.05, FTH1P3 vs. blank control; P<0.001 si-FTH1P3 vs. si-control; Fig. 1D). The results of MTT and BrdU assays indicated that overexpression of FTH1P3 promoted glioma U251 cell proliferation. In addition, flow cytometry revealed that the percentage of apoptotic cells in the pc-FTH1P3 group was significantly lower compared with that of the blank group, whereas the percentage in the siRNA-FTH1P3 group was significantly increased compared with the siRNA-control group (P<0.01, FTH1P3 vs. blank control; P<0.01 si-FTH1P3 vs. si-control; Fig. 1E). Notably, western blot analysis demonstrated that the alterations in BAX/BCL2 expression were in line with the percentage of apoptotic cells in each transfected group (P<0.05, FTH1P3 vs. blank control; P<0.001 si-FTH1P3 vs. si-control; Fig. 1F). These data indicated that overexpression of FTH1P3 may have inhibited glioma U251 cell apoptosis via regulation of BAX/BCL2 expression.

It has been reported that FTH1P3 facilitates the progression of oral squamous cell carcinoma by inhibiting miR-224-5p (16). Thus, the regulatory association between FTH1P3 and miR-224-5p in U251 cells was investigated. As presented in Fig. 2A, miR-224-5p expression was significantly decreased in glioma tissues compared with normal tissues (P<0.05); however, there was no significant difference in miR-224-5p expression between high- and low-grade glioma tissues. In addition, miR-224-5p expression was significantly decreased in the pc-FTH1P3 group and increased in the siRNA-FTH1P3 group, compared with the corresponding control groups (P<0.01, FTH1P3 vs. blank control; P<0.05 si-FTH1P3 vs. si-control; Fig. 2B). These data indicated that miR-224-5p expression was inhibited by FTH1P3 in glioma U251 cells. To further examine the functional role of miR-224-5p in glioma U251 cells, miR-224-5p was overexpressed. As presented in Fig. 2C, the results demonstrated that the expression of miR-224-5p was significantly increased by transfection with miR-224-5p mimic, indicating a high transfection efficiency (P<0.01).

To further examine the downstream regulatory mechanism of miR-224-5p, the target genes of miR-224-5p were predicted by TargetScanHuman. The results revealed that TPD52 was a potential target of miR-224-5p (Fig. 3A). The luciferase reporter assay demonstrated that miR-224-5p mimic decreased the luciferase activity of TPD52 3′-UTR-WT (P<0.05), although not that of TPD52 3′-UTR-MUT (Fig. 3B). In addition, the mRNA and protein expression of TPD52 was significantly decreased following overexpression of miR-224-5p (P<0.05; Fig. 3C and D). These data indicated that TPD52 was a target of miR-224-5p, and TPD52 expression was negatively regulated by miR-224-5p.

The expression of TPD52 in glioma tissues was further determined by western blot analysis. The results revealed that TPD52 expression in glioma tissues was significantly increased compared with normal tissues, with the highest expression in high-grade glioma tissues (P<0.05; Fig. 4A). To further analyze the functional role of TPD52, U251 cells were transfected with pcDNA-TPD52. The results demonstrated that the protein expression of TPD52 was significantly increased by pcDNA-TPD52 (P<0.001; Fig. 4B). Additionally, compared with the mimic control group, TPD52 expression was significantly downregulated in U251 cells following transfection with miR-224-5p mimic. This effect was markedly reversed following co-transfection of miR-224-5p mimic and pc-TPD52 (P<0.01; Fig. 4C). Furthermore, the results of the MTT and BrdU assays revealed that cell viability and the number of BrdU-positive cells was significantly inhibited following miR-224-5p overexpression. These effects were also markedly reversed following co-transfection with miR-224-5p mimic and pc-TPD52 (P<0.05, at 24 h; P<0.01, at 48 h and P<0.01 at 72 h; Fig. 4D and E). Finally, miR-224-5p mimic transfection significantly induced cellular apoptosis and increased the expression of BAX/BCL2 in U251 cells, which was significantly reversed following co-transfection of miR-224-5p and TPD52 (P<0.01; Fig. 4F and P<0.001 miR-224-5p mimic vs. mimic control, P<0.01, miR-224-5p mimic+TPD52 vs. miR-224-5p mimic + blank control; Fig. 4G). Taken together, these data indicated that FTH1P3 may act through the miR-224-5p/TPD52 axis in glioma (Fig. 4H).