Samples of human glioma and adjacent healthy tissues were obtained from 20 patients (11 male and 9 female, age, 44.6±18.4) undergoing surgery at the Department of Neurosurgery, Nanfang Hospital of Southern Medical University (Guangzhou, China). None of the patients had received prior chemotherapy or biotherapy, and diagnoses were confirmed pathologically in all cases. Central glioma tissues and adjacent nontumor tissues, located 2 cm from the center in the same patient were collected. All specimens were snap frozen at the time of surgery and stored in liquid nitrogen. The samples were collected with informed consent from patients and ethical consent was granted from the Committees for Ethical Review of Research involving Human Subjects of Southern Medical University.

Total RNA from the cultured cells or human tissues was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance with the manufacturer's protocol. Prior to the addition of TRIzol, the tissues were ground using liquid nitrogen. The miRNAs involved in putatively regulating NFIA were detected using the All-in-One™ miRNA RT-qPCR kit (GeneCopoeia, Rockville, MD, USA) according to the manufacturer's protocol in 20 µl reaction volumes. Prior to the addition of TRIzol, the tissues were ground with liquid nitrogen. cDNA was generated from 1000 ng of total RNA and 2 µl cDNA were used for PCR analysis. The amplification conditions were as follows: Stage 1, 95°C for 30 sec; stage 2, 95°C for 3 sec and then 60°C for 30 sec, 40 cycles; stage 3, 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec, 60°C for 15 sec. The RT-qPCR was performed on an ABI 7500 Fast Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression of U6 RNA was used as an endogenous control (28). The mRNA levels were evaluated using the ABI 7500 Fast Real-Time PCR system with SYBR green detection chemistry (Takara Bio, Inc., Shiga, Japan). The expression of GAPDH was used as an internal control (29). Quantitative measurements were determined using the ΔΔCq method (30). All samples were measured in triplicate and the mean value was considered for comparative analysis. The sequences of the primers used are listed in Table I.

U251 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained according to the supplier's recommendation. The U251 human glioma cells were maintained in Dubecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) with high glucose and sodium pyruvate, supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). The cells were maintained in a humidified incubator at 37°C with 5% CO2.

Packed empty lentivirus (LV) vectors (Landbiology, Guangzhou, China) with green fluorescent protein (GFP; LV-Mock), an LV-mediated lncRNA RP5-833A20.1-overexpression vector (LV-RP5-833A20.1), and an LV-mediated lncRNA RP5-833A20.1-knockdown vector (LV-siRNA-RP5-833A20.1) with GFP were prepared as described previously (31). The U251 cells were incubated in 6-well plates for 24 h at a density of 2ⅹ10⁶ cells/well and then were transfected using DMEM and polybrene reagents (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) according to the manufacturer's protocol at a multiplicity of infection of 1. The U251 human glioma cells were maintained in Dubecco's modified Eagle's medium (DMEM) with high glucose and sodium pyruvate, supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), and plus 2 µg/ml puromycin for 2 weeks to gain stable clones. The cells were maintained in a humidified incubator at 37°C with 5% CO₂. The expression level of RP5-833A20.1 was determined using RT-qPCR analysis.

For the cell proliferation assays, the U251 cells were seeded in 96-well plates at the density of 2×10³ cells/well. Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) solution (10 µl) was added to each well and the cells were incubated for 1.5 h at 37°C. The absorbance at 450 nm was recorded using a Thermo Multiskan MK3 reader (Thermo Fisher Scientific, Inc.). A total of five replicate wells were set up in each group and five independent experiments were performed.

For the invasion assays, 5×10⁴ cells in 200 ml serum-free medium were seeded into the upper chamber of a Transwell insert (8-mm pore size; EMD Millipore, Billerica, MA, USA) coated with Matrigel. The lower chamber was filled with 600 liters medium containing 10% FBS. Following incubation in a humidified incubator at 37°C with 5% CO₂ for 12 h, the cells remaining on the upper membrane were removed with a cotton swab. The cells, which had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet, images were captured, and the cells were counted using a BX51 microscope (Olympus, Tokyo, Japan). Invasion was assessed by counting the number of stained cell nuclei from 10 randomly selected fields per filter in each group (magnification, ×400), with the cell counts expressed as the mean number of cells per field of view. Three independent experiments were performed in triplicate.

The U251 cells were harvested 48 h following transfection and resuspended 100 µl of the solution (1 × 10⁵ cells) to a 5 ml culture tube and add 5 µl of FITC Annexin V (BD Biosciences, San Jose, CA, USA) and 5 µl PI (BD Biosciences). Next, the cells were gently vortexed and incubated for 15 min at 25°C in the dark, 400 µl of 1X Binding Buffer was added to each tube. The results were analyzed using a flow cytometer (FACScan; BD Biosciences) within 1 h. Cell Quest version 6.0 (BD Biosciences) was used to analyze the results. All samples were assayed in triplicate.

The treated U251 cells were trypsinized, fixed in 70% ethanol, washed twice with phosphate-buffered saline (PBS), and then labeled with propidium iodide (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) in the presence of 30 KU/ml RNase A (KeyGen Biotech Co., Ltd.) for 30 min in the dark (50 g/ml). The samples were run on a FACScan flow cytometer (BD Biosciences), and the percentages of cells within each phase of the cell cycle were analyzed using Cell Quest version 6.0.

The cells were washed twice with ice-cold PBS and the cell lysates were harvested using a KeyGen Biotech Whole Cell Lysis Assay kit (KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. The proteins were quantified by KeyGEN Bradford protein quantitation assay (KeyGen Biotech Co., Ltd.) and 50 µg protein per lane was fractionated by 12% SDS-PAGE, transferred onto a PVDF membrane, blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Haimen, China) for 1 h at room temperature and immunoblotted with primary antibodies overnight at 4°C. Following incubation with the corresponding secondary antibodies conjugated to horseradish peroxidase, the signals of the membranes were detected by chemiluminescence western blotting substrate (Thermo Fisher Scientific, Inc.). The band intensity of western blotting and the normalization were analyzed using ImageJ version 1.6.0 software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used were obtained from Abcam (Cambridge, UK) as follows: NFIA (cat. no. ab41851; 1:1,000); p53 (cat. no. ab31333; 1:1,000); p21 (cat. no. ab47452; 1:1,000); PAI-1 (cat. no. ab66705; 1:1,000) and β-actin (cat. no. ab8227; 1:2,000). The secondary antibody used was goat anti-rabbit IgG (cat. no. bs-0295G; 1:5,000; BIOSS, Beijing, China).

To assess the methylation level of the NFIA gene promoter, U251 cells with knocked down RP5-833A20.1 or transfected with a short hairpin negative control vectors (LAND) were used. Genomic DNA was isolated using a Promega wizard genomic DNA purification kit (Promega, Madison, MA, USA) according to the manufacturer's protocol. DNA modification was performed using an EpiTect Bisulfite kit (Qiagen, Shanghai, China) to convert unmethylated cytosines to uracils. The PCR was performed using the aforementioned protocol. Following BSP, the PCR products were used for cloning and sequencing. The sequences of the BSP primers are listed in Table I.

All experiments were performed three times. Data are presented as the mean ± standard deviation and analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) with Student's t-test or one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

In our previous study, it was shown that the RP5-833A20.1/miR-382-5p/NFIA pathway is essential for the regulation cardiovascular disease (27). Several studies have shown that NFIA is important in glioma (25,26). Thus, the present study hypothesized that RP5-833A20.1 may also function in glioma by regulating the expression of NFIA. To confirm this, the present study examined the RNA expression levels of RP5-833A20.1 and NFIA in 20 paired glioma and adjacent nontumor tissue samples. The expression of RP5-833A20.1 was significantly downregulated (P<0.01) in 80% (16/20) of the cancerous tissues, compared with the paired adjacent nontumor tissues (Fig. 1A). The expression of NFIA was significantly upregulated (P<0.01) in 90% (18/20) of the cancerous tissues, compared with the paired adjacent nontumor tissues (Fig. 1B).

In our previous study, it was found that RP5-833A20.1 suppressed the expression of NFIA by promoting the expression of miR-382-5p in THP-1 macrophages (27). The present study examined whether RP5-833A20.1 regulates the expression of NFIA by promoting miR-382-5p in U251 cells. Lentivirus-mediated overexpression of RP5-833A20.1 in the U251 cells(Fig. 2A) markedly inhibited the mRNA and protein expression levels of NFIA (Fig. 2B and C), whereas overexpression of RP5-833A20.1 increased the expression of miR-382 in the U251 cells (Fig. 2D). These results indicated that RP5-833A20.1 suppressed the expression of NFIA in the U251 cells and that miR-382-5p may be involved in this process.

Increasing evidence has confirmed that lncRNAs can regulate the DNA methylation of protein-coding genes during the development of disease (11,32,33). To assess the role of RP5-833A20.1 in the regulation of DNA methylation, the present study assessed the methylation level of the NFIA gene promoter using BSP. As shown in Fig. 3A and B, the knock down of RP5-833A20.1 decreased the methylation level of the NFIA promoter in the U251 cells, compared with the control (3.8, vs. 9.5%, respectively; P<0.05). These results indicated that RP5-833A20.1 repressed the expression of NFIA by enhancing the methylation level of the NFIA promoter.

As RP5-833A20.1 was downregulated in glioma tissues and suppressed the expression of NFIA, the present study then investigated the effect of RP5-833A20.1 on cell proliferation and invasion. CCK-8 assays revealed that LV-RP5-833A20.1 treatment significantly decreased the proportion of living U251 cells (Fig. 4A). The invasive capacity of the cells was investigated using Transwell assays. Compared with the control groups, the overexpression of RP5-833A20.1 inhibited the migratory ability of the U251 cells (Fig. 4B and C).

The present study also investigated whether RP5-833A20.1 is involved in cell cycle distribution and apoptosis. FACS analysis showed a significant decrease and increase in the numbers of cells in the S and G1 phases, respectively, in the U251 cells infected with LV-RP5-833A20.1, compared with the control (Fig. 5A and B). Consistent with the FACS data, the expression of G1/S phase checkpoint proteins, including tumor suppressor p53 (P53) (34), cell cycle regulator p21 (P21) (35) and plasminogen activator inhibitor 1 (PAI-1) (36) were markedly upregulated in the cells infected with LV- RP5-833A20.1 (Fig. 5C). To determine whether RP5-833A20.1 has a function in cell apoptosis, flow cytometry was performed. As shown in Fig. 5D, LV- RP5-833A20.1 significantly induced the apoptotic rate of the U251 cells.