A total of 80 patients with diagnosed glioma (Male: Female, 59:21, age) were enrolled in the present study. For all patients, the dissected glioma tissues and their adjacent noncancerous tissues were collected from Tianjin First Center Hospital (Tianjin, China) during January 2014 to December 2016. All of the tissues were frozen in liquid nitrogen following dissection from patients during surgeries. Clinical data were also recorded for statistical analysis. Each patient provided informed consent to participate in the present study, which was approved by the Ethics Committee of Tianjin First Center Hospital.

Human glioma cell lines BE-2C and BT325 were purchased from the American Type Culture Collection (Manassas, VA, USA). Human glioma cell line SWO38 was a kind gift from Jinan University (Guangzhou, China) (18). Human glioma cell lines U251-MG, SHG-44 and CHG-5 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). Human normal neuronal cell line. Human normal astrocyte cells were purchased from ScienCell Research Laboratories, Inc. (San Diego, CA, USA; catalog no. 1830) and used as a normal control of glioma cell. All culture media (Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were supplied with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Small interfering (si)RNA against TSLNC8 was designed and synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd., (Shanghai, China) with the sequence of 5′-GCACATGAACACATTGAAA-3′ and the control siRNA sequence was 5′-GCAAAGTACACGTTACAAA-3′ with the same source as the specific siRNAs. TSLNC8 expressing plasmid was constructed by our own lab with XhoI and HindIII restriction enzyme and cloned into pcDNA 3.1 vector. A total of 2 µg siRNAs or expressing plasmid was transfected into cells with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 12 h, the cell culture medium was replaced with new one. In all conditions, the culture medium was replaced every 2 days, unless otherwise stated.

Total RNA from human tissues and all cultured cells was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). RNA was quantified by Nanodrop 2000 (Thermo Fisher Scientific, Inc., Wilmington, DE, USA) by measuring the optical density at a wavelength of 260 nm. RT was then immediately performed using Prime Script TM Master Mix (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Subsequently, RT-qPCR was performed with SYBR^® Premix EX Taq TM II (Takara Bio, Inc.) on the real-time PCR detection system ABI7900 (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as the internal reference, and gene mRNA expression levels were calculated using the 2^−ΔΔCq method (19). The thermocycling protocol was conducted as follows: Initial denaturation at 95°C for 5 min, followed by 45 cycles of a three-step cycling program consisting of 10 sec at 95°C (denaturation), 10 sec at 60°C (primer annealing) and 10 sec at 72°C (elongation), and a final extension step for 10 min at 72°C. The primer sequences used for qPCR were as follows: TSLNC8 forward, 5′-TGATCCTCATAGTATAATG-3′ and reverse, 5′-AGTTCTTTAGCAGTACATG-3′; GAPDH forward, 5′-GTGGACATCCGCAAAGAC-3′ and reverse, 5′-AAAGGGTGTAACGCAACTA-3′.

Cell viability was determined via an MTT assay. A total of 1,000 U251-MG and SWO38 cells were transfected with TSLNC8-overexpressing plasmid and SHG-44 and BT325 cells were treated with siRNAs with or without TSLNC8 knockdown for 48 h, trypsinized and reseeded in triplicate in 96-well plates at an initial density of 4,000 cells per well. Cell numbers were monitored for a total of 5 consecutive days. At each indicated time points (days 1, 2, 3, 4 and 5), cell cultures were incubated with 10 µl MTT solution (5 mg/ml, Beyotime Institute of Biotechnology, Haimen, China) per well for 2 h at room temperature. The absorbance was recorded at a wavelength of 570 nm. Cell viability was defined as the cell number ratio of experimental groups to control cells.

For cell migration assays, U251-MG and SWO38 cells were transfected with TSLNC8-overexpressing plasmid for 48 h and then trypsinized and collected by low-speed centrifugation (1,000 × g, 4°C for 5 min) with serum-free DMEM. A total of 1×10⁴ cells (~150 µl) were spread into the upper chamber. The lower chamber was filled with 600 µl DMEM supplemented with 10% FBS. Subsequently, the plate was incubated for 24 h at 37°C in an incubator and the cells were allowed to grow freely. At 24 h post-seeding, the membrane was fixed with pre-cooled methanol at room temperature for 10 min and stained with crystal violet (1%) for 5 min at room temperature. Cell migration was assessed by counting the cells that had migrated through the membrane. A total of 5 random fields were selected and images captured under a Nikon light microscope (Nikon Corporation, Tokyo, Japan) at a magnification of ×100. For cell invasion assays, the membrane was pre-coated with Matrigel (Corning Incorporated, Corning, NY, USA) for 6 h in a 37°C incubator.

U251-MG and SWO38 cells were transfected with TSLNC8-overexpressing plasmid for 48 h and were then cultured in DMEM in a 6-well culture plate at a density of 5×10⁵ cells/well overnight until 90% confluence was attained. The culture medium was replaced with serum-free DMEM. A line was scratched in the single cell layer using a 10 µl pipette tip and the cells were then washed with PBS three times. Following incubation at 37°C for 24 h, images of the migrated cells were observed and images were captured using a Nikon light microscope (magnification, ×200).

The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay was performed according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, a total of 10,000 U251-MG and SWO38 cells were plated into 6-well plates and transfected with TSLNC8-overexpressing plasmid for 48 h; SHG-44 and BT325 cells were transfected with control or specific siRNA against TSLNC8. Subsequently, cells were washed with pre-cooled PBS, trypsinized, and re-suspended in 100 µl of binding buffer with 2.5 µl FITC-conjugated Annexin V and 1 µl PI (100 µg/ml). Cells were then incubated at room temperature for 15 min in darkness. A total of ≥10,000 cells were collected and analyzed.

The activities of caspase-3, −8 and −9 were determined using the caspase activity kits (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's protocols. Briefly, U251-MG and SWO38 cells were plated into 6-well plates and transfected with TSLNC8-overexpressing plasmid for 48 h; SHG-44 and BT325 cells were transfected with control or specific siRNA against TSLNC8. Subsequently, cell lysates were collected by low speed centrifugation (1,000 × g, for 5 min at 4°C). A total of 10 µl protein from each sample were added into 96-well plates and mixed with an aliquot of 80 µl reaction buffer supplied with caspase substrates (2 mM). Following incubation at 37°C for 4 h, caspase activities were determined with a Tecan reader (Tecan Group, Ltd, Mannedorf, Switzerland) at an absorbance of 450 nm.

GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA) software was used for statistical analysis. Data are presented as the mean ± standard deviation. The two-tailed Student's t-test was used to compare means of two groups, while one-way analysis of variance was used for comparisons among multiple groups (≥3 groups), followed by a Least Significant Difference post hoc test. The Spearman correlation analysis was used to evaluate the experimental data in Table I. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated at least three times.

First, the relative transcript levels of TSLNC8 in 80 glioma patients were analyzed in the present study. As presented in Fig. 1A, the expression of TSLNC8 was significantly decreased in 95% cases of glioma patients (76 cases) compared with in adjacent noncancerous tissues (P<0.01). Of note, TSLNC8 transcript levels higher than the median level were characterized as high TSLNC8 expression (n=37); transcript levels lower than the median level were characterized as low TSLNC8 expression (n=43). The clinical data were also analyzed and presented in Table I, which indicated that the relative transcript levels of TSLNC8 were negatively associated with tumor size (P=0.001), distant metastasis (P=0.004) and TNM stage (P=0.003), and not associated with age (P=0.2445), sex (P=0.960) and lymph node metastasis (P=0.155). Then, five glioma cell lines, including U251-MG, SHG-44, BT325, SWO38, CHG-5 and normal astrocyte cell line used as a control were analyzed. RT-qPCR analysis revealed that all of the five glioma cell lines exhibited significantly lower transcript levels of TSLNC8 compared with in the control astrocyte cells (Fig. 1B), of which U251-MG and SWO38 demonstrated the lowest expression levels of TSLNC8. Additionally, U251-MG and SWO38 cells exhibited the highest potential to migrate; SHG-44 and BT325 revealed the highest expression of TSLNC8 of the 7 glioma cell lines and the migration capacities of these cell lines were the lowest (Fig. 1B). Therefore, U251-MG and SWO38 were selected for the knockdown experiments and SHG-44 and BT325 were used for overexpression studies. All of these data showed that the transcript level of TSLNC8 was decreased in human glioma in vivo and in vitro.

To investigate the role of TSLNC8 in human glioma, TSLNC8 was overexpressed in U251-MG and SWO38 cells and downregulated in SHG-44 and BT325 cells using an overexpression plasmid or siRNAs, respectively. When U251-MG and SWO38 cells were transfected with TSLNC8-overexpression plasmid, the transcript levels of TSLNC8 were significantly increased by 4.5- and 4-fold, respectively (Fig. 2A). In addition, the expression levels of TSLNC8 in SHG-44 and BT325 cells were significantly decreased by >50% of the original levels upon transfection with siTSLNC8 (Fig. 2B). Subsequently, the proliferation rate of the 4 glioma cell lines was analyzed. No significant alterations in the first 2 days in experimental cells were observed; however, cell proliferation decreased by 17% on day 4 and 19% on day 5 of U251 cells transfected with TSLNC8-expressing plasmid (Fig. 2C). Furthermore, SWO38 cell proliferation rate decreased on days 4 and 5 with TSLNC8 overexpression (Fig. 2D); the cell proliferation rate increased on days 4 and 5 in SHG-44 and BT325 cells transfected with siRNA against TSLNC8 (Fig. 2E and F, respectively). These results suggested that TSLNC8 may suppress cell proliferation in human glioma cells.

As well as cell proliferation rate, the effects of TSLNC8 on cell metastasis were investigated. To this end, TSLNC8-expressing plasmid was transfected into U251-MG and SWO38 cells for 48 h. Transwell assays revealed that >400 U251 and SWO38 cells were observed to migrate via the membrane; however, only ~200 cells were counted on the lower side of the membrane upon transfection with TSLNC8-expressing plasmid (Fig. 3A and B). Additionally, cell invasion was also inhibited by TSLNC8 overexpression in both U251-MG and SWO38 cells (Fig. 3A and C). Subsequently, wound-healing assays were also performed in U251-MG and SWO38 cells. As presented in Fig. 3D and E, the wound closure ability of U251-MG cells was inhibited by >50% when cells were treated with TSLNC8-overexpression plasmid. Furthermore, the wound closure ability of SWO38 cells was also inhibited by 50% upon TSLNC8 overexpression (Fig. 3D and F). These data suggested that TSLNC8 suppressed cell metastasis in human glioma.

Finally, cell apoptosis was assessed in glioma cells. As presented in Fig. 4A, cell apoptosis was increased by 2.7-fold in U251-MG cells and 2.5-fold in SWO38 cells of the TSLNC8 treated groups compared with in the control cells; the cell apoptotic rate of SHG-44 and BT325 cells were significantly decreased by >50% upon siTSLNC8 stimulation (Fig. 4B). Subsequently, the relative caspase activities were analyzed. Treatment with TSLNC8-expressing plasmid of U251-MG and SWO38 cells increased, while stimulation of siTSLNC8 in SHG-44 and BT325 cells decreased the activities of caspase-3, respectively (Fig. 4C and D). Interestingly, the activity of caspase-8 remained stable upon transfection with TSLNC8-expressing plasmid or siTSLNC8 in glioma cells (Fig. 4E and F). The relative activities of caspase-9 were also significantly altered, following similar trends to those exhibited by caspase-3, in the different glioma cell lines (Fig. 4G and H). As caspase-3 and −9 are key factors involved in the intrinsic apoptosis pathway and caspase-8 serves significant roles in the extrinsic apoptotic pathway (20), the findings of the present study suggested that TSLNC8 may promote cell apoptosis via the intrinsic pathway in human glioma.