The human GBM cell lines U87-MG (glioblastoma of unknown origin; cell line was authenticated by STR profiling) and T98G (purchased in 2014 from the Cell Bank of the Chinese Academy of Sciences) were cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained in a humidified atmosphere at 37°C with 5% CO₂.

Cells in culture were treated with an irradiator (GE 3000; GE Healthcare Life Sciences) using a ¹³⁷Cs source at an exact dose of 0 or 6.0 Gy. During irradiation, the cultures were stored in the cell culture incubator (5% CO₂ at 37°C). The cells were harvested exactly at the end of the irradiation.

Reverse transcription-quantitative PCR. Total RNA was extracted from cellular samples using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. UV spectrophotometry was used to determine the RNA concentration and quality. Reverse transcription of total RNA was performed using an iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer's instructions. A 7500 Fast PCR instrument (Applied Biosystems, Thermo Fisher Scientific, Inc.) was used for quantitative PCR amplification. The primer and probe sequences were as follows: LGMNP1 forward primer, 5′-GGACGTGGAAGATCTGACTAACC-3′, reverse primer, 5′-ATGATGTGGCTGGTATTGGTGTAT-3′ and probe, 5′-VIC-CAAGCAGTGCCGCC-MGB-3′. The probe was modified with MGB at the 3′-end. GAPDH forward primer, 5′-GAAGGACTCATGACCACAGTCCA-3′, reverse primer, 5′-GCAGGGATGATGTTCTGGAGAG-3′ and probe, 5′-ROX-CGGCCATCACGCCACAGTTTCC-3′-BHQ2. Gene alignments and primer specificity analysis were used to choose specific primers and probes. The composition of the reaction mixture contained 10 µl of 2X TaqMan Universal Master Mix II with UNG (Applied Biosystems, Thermo Fisher Scientific, Inc.), 1 µl (100 ng) of cDNA, 1 µl of probe-primer mix and 8 µl of nuclease-free water, constituting to a final volume of 20 µl. The thermocycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min and 45 cycles of 95°C for 15 sec and 60°C for 1 min. Data were acquired at the end of the annealing/extension phase. Melting curve analysis was performed at the end of each run from 50 to 95°C.

The LGMNP1 overexpression sequence was constructed by Shanghai Hanyin Co., Ltd. (Shanghai, China). The recombinant lentivirus and negative control (NC) lentivirus were prepared and titered to 10⁹ transfection U/ml. After 48 h, the efficiency of overexpression was confirmed via RT-qPCR. To obtain stably transfected cells (LGMNP1-OE), GBM cells were seeded in 6-well dishes at a density of 1×10⁵ cells/well. The cells were then infected with the same virus titer on the following day with 8 µg/ml Polybrene. At 72 h post-viral infection, the culture medium was replaced with selection medium containing 4 µg/ml puromycin. The puromycin-resistant cells were amplified in medium containing 2 µg/ml puromycin for 7 days and then transferred to medium without puromycin.

The isolated cells were seeded at 300 cells/well in a 6-well tissue culture plate and grown for 14 days until macroscopic cell clones were visible. The cells were then fixed with 95% cold methanol for 15 min at 4°C and stained with 0.5% methylene blue for 2 min in order to determine the number of colonies by microscopy. The colony forming efficiency was calculated as the percentage of single cells that generated colonies on the 14th day. The colony formation rate was calculated as follows: Colony formation rate = (number of clones/300) × 100%.

Cells were lysed by placing the slides in a Coplin jar (Thomas Scientific, Swedesboro, NJ, USA) containing 2.5 M NaCl, 0.1 M Na₂EDTA, 0.1 M Tris and 1% Triton X-100 (pH 10) at 4°C for at least 1 h. Subsequently, slides were immersed in electrophoresis solution (0.3 M NaOH and 1 mM Na₂EDTA, pH >13) for 30 min. Electrophoresis was then performed at 1.3 V/cm for 20 min in the same solution. Slides were washed twice in cold phosphate-buffered saline (PBS) for 10 min and in water for another 10 min. Comets were fixed by immersing the slides in 70% ethanol for 15 min and in absolute ethanol for a further 15 min prior to placing them on the bench to dry overnight. Comets were stained with SYBRGold at the dilution recommended by the manufacturer in a bath at 4°C with agitation. After 40 min, SYBRGold solution was removed and the slides were rinsed twice with water and left to dry at room temperature. On the day of analysis, gels were hydrated by adding a drop of water on top of each minigel, and a glass coverslip (24×60 mm) was used to cover all the minigels on the slide. The semi-automated image analysis system Comet Assay IV (Perceptive Instruments Ltd., Bury St. Edmunds, UK) was used to evaluate 100 comets/gel in the case of the H₂O₂ experiments. Percentage DNA in the tail was the parameter selected to describe each comet. The number of comets per gel (including so-called hedgehogs) was counted by direct observation.

Apoptosis was analyzed by translocation of phosphatidylserine to the cell surface using an Annexin and DAPI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were treated with 6 Gy of radiation, then collected and washed in cold PBS. Cells were resuspended in Annexin V-FITC and DAPI for 30 min in the dark. Cell apoptosis was analyzed on a FACSAria flow cytometer (BD Biosciences) and quantified using CellQuest software 5.1 (BD Biosciences). Fluorescence was captured with an excitation wavelength of 480 nm.

The total proteins of cells were extracted with cell lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40 and 1 mM PMSF) and determined by BCA methods. Protein samples (30 µg) were subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with blocking buffer [5% skimmed milk in Tris-buffered saline containing Tween-20 (TBS-T)] at room temperature for 1 h. The membranes were then incubated with the following antibodies at a 1:500 dilution at 4°C overnight: caspase-3 antibody (cat. no. 9662), caspase-7 antibody (cat. no. 12827), Bax antibody (cat. no. 14796; all from Cell Signaling Technology, Inc., Danvers, MA, USA), active caspase-3 antibody (cat. no. ab2302; Abcam, Cambridge, UK) and β-actin antibody (cat. no. 4970; Cell Signaling Technology, Inc.). The membranes were washed with TBS-T, then incubated with horseradish peroxidase-conjugated anti-rabbit (cat. no. R2655) or anti-mouse antibody (cat. no. M8270; both from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a 1:10,000 dilution at room temperature for 2 h. Detection was performed using western blot detection reagents (Odyssey; LI-COR Biosciences, Lincoln, NE, USA).

Values are expressed as the mean ± standard error of the mean. One-way analysis of variance (ANOVA) with Tukey's post hoc test was performed for comparison of multiple groups. All statistical analyses were performed using SPSS for Windows v. 17.0 (SPSS, Inc., Chicago, IL, USA). A two-tailed P<0.05 was considered to indicate a statistically significant difference.

To determine the potential role of LGMNP1 in GBM radiotherapy resistance, RT-qPCR was employed to detect the relative expression of LGMNP1 in two glioma cell lines after exposure to a radiation dose of 6 Gy compared with that in the control group (0 Gy) (Fig. 1A and B). The result indicated that in each of the two cell lines, the relative expression of LGMNP1 in the experimental group was significantly higher than that in the control group (P<0.0001). It was, therefore, indicated that the expression of LGMNP1 is associated with radiotherapy.

To verify the function of LGMNP1 in radiotherapy resistance of GBM, U87-MG and T98G cells with stable overexpression of LGMNP1 were established using the lentiviral vector-mediated method. As is presented in Fig. 2A and B, LGMNP1 was effectively upregulated in LGMNP1-OE cells compared with that in the NC cells. The clonogenic assay indicated that the colony formation ability of LGMNP1-OE cells after radiotherapy was greater than that of the NC cells (Fig. 3). These results suggested that overexpression of LGMNP1 contributes to radiation resistance.

One of the mechanisms by which radiotherapy kills tumor cells is the induction of DNA double-strand breaks. In order to determine whether overexpression of LGMNP1 improves the capacity for DNA damage protection after exposure to radiation damage, the cell lines were subjected to the comet assay. The results indicated that the percentage of tail DNA was higher in the cells exposed to 6 Gy of radiation compared with that of cells that received 0 Gy (Fig. 4). Furthermore, the percentage of tail DNA in the LGMNP1-OE group was less than that in the NC group, in the T98G cell line (Fig. 4A and B) and also in the U87-MG cell line (Fig. 4C and D). These results suggested that overexpression of LGMNP1 improves the ability of glioma cells to perform DNA damage protection. To detect apoptosis in glioma cell lines, flow cytometric analysis was employed, demonstrating a lower ratio of apoptosis in LGMNP1-OE vs. NC glioma cells after radiotherapy (Fig. 5). For further verification, the levels of apoptotic proteins were assessed by western blot analysis. The results revealed that the levels of apoptotic proteins, including caspase-3, active caspase-3, caspase-7 and Bax in LGMNP1-OE glioma cells treated with radiotherapy were decreased compared with those in the NC group, and almost the same results were obtained with the two cell lines (Fig. 6). Overall, these results indicated that overexpression of LGMNP1 in GBM cell lines enhances the capacity for DNA damage protection and reduces apoptosis following radiation-induced damage.