The present study examined clinical data from 66 patients diagnosed with PC and treated at Tongji Hospital (Wuhan, China) between January 2011 and January 2013. Prostate biopsy tissues and serum samples were obtained from all patients. The inclusion criteria were as follows: i) Patients were diagnosed with PC by pathological examinations; ii) patients completed the whole treatment procedure at Tongji Hospital; iii) patients completed the follow-up and had complete clinical data record; and iv) patients received no treatment before admission. The exclusion criteria were as follows: i) At the time of the study, patients suffered or had suffered from other prostate diseases; ii) at the time of the study, patients suffered or had suffered from another cancer; iii) patients who were treated before admission; iv) patients who failed to complete the whole treatment procedure; and v) patients who died of other causes during the follow up. Prostate biopsy tissues and serum samples were also collected from 56 healthy subjects who received physiological examinations at Tongji Hospital between January 2011 and January 2013. These subjects underwent prostate biopsy to detect potential prostate lesions; prostate diseases were eventually excluded. The age of patients in the PC group ranged between 47 and 86 years (age, 68.9±5.1 years), and those in the healthy Control group ranged between 46 and 85 years (age, 69.1±5.0 years). No significant differences in age, smoking and drinking habits, and other basic clinical data, including body mass index and height, were identified between these two groups. The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology approved the present study. Written informed consents were obtained from all participants and/or their families.

The HPrEC human normal prostate epithelial cell line [cat. no. PCS-440-010™; American Type Culture Collection (ATCC), Manassas, VA, USA] and the two human PC cell lines 22Rv1 (cat. no. CRL-2505™; ATCC) and DU145 (cat. no. HTB-81™; ATCC) were used in the present study. All cells were cultured with RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂.

An EcoRI-EcoRI fragment containing full length GASL1 cDNA was amplified by polymerase chain reaction (PCR), and a GASL1 expression vector was constructed by inserting this fragment into the pIRSE2-EGFP vector (Clontech Laboratories, Inc., Mountainview, CA, USA). GASL1 expression vector or empty pIRSE2-EGFP vector (10 nM) was transfected into 6×10⁵ cells using Lipofectamine^® 2000 reagent (cat. no. 11668-019; Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Incubation with vectors was performed at 37°C for 6 h. Subsequent experiments were performed 12 h after transfections. Expression of GASL1 was detected prior to and following subsequent experiments, and an overexpression rate >150% (150–280%) was reached every time.

Following transfection, HPrEC, 22Rv1 and DU145 cells were collected, washed with PBS and resuspended in RPMI-1640 medium (catalog no. 30-2001; ATCC) to the density of 3×10⁴/ml. Cell proliferation was measured by CCK-8 assay. The highly water-soluble tetrazolium salt WST-8 is reduced by cellular dehydrogenases, which becomes orange in color (formazan); the amount of formazan is proportional to the number of living cells. Cells were cultured in a 96 well plate at a density of 3×10³ cells/100 µl/well and placed in the incubator. CCK-8 solution (10 µl; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added at 24, 48, 72 and 96 h. Cells were then cultured for additional 4 h and 10 µl DMSO was added. OD values were measured at 450 nm using Fisherbrand™ accuSkan™ GO UV/Vis microplate spectrophotometer (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate. At 24, 48, 72 and 96 h, cells were subjected to RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to confirm the overexpression of GASL1.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific Inc.) was used to extract total RNA from HPrEC, 22Rv1 and DU145 cells (3×10⁴ cells/ml). To achieve complete cell lysis, tissues (0.05 g) were ground in liquid nitrogen prior to addition of TRIzol. RT was performed to synthesize cDNA using SuperScript IV Reverse Transcriptase kit (Thermo Fisher Scientific, Inc.) with the following conditions: 25°C for 6 min, 55°C for 20 min and 80°C for 10 min. qPCR reaction systems were prepared using SYBR^® Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, Inc.). All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR primers sequences were as follows: GASL1, forward 5′-CTGAGGCCAAAGTTTCCAAC-3′, reverse 5′-CAGCCTGACTTTCCCTCTTCT-3′; and β-actin, forward 5′-GACCTCTATGCCAACACAGT-3′, reverse 5′-AGTACTTGCGCTCAGGAGGA-3′. Thermocycling conditions were as follows: 95°C for 50 sec, followed by 40 cycles of 95°C for 15 sec and 60°C for 35 sec. Expression of GASL1 was normalized to β-actin endogenous control using the 2^−ΔΔCq method (12). This experiment was performed in triplicate.

Total protein was extracted from HPrEC, 22Rv1 and DU145 cell lines 12 h after transfection using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific Inc.) with a cell density of 3×10⁴ cells/ml. The bicinchoninic acid assay was used to measure protein concentration. Proteins (30 µg per lane) were separated by 10% SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% skimmed milk at room temperature for 1 h. Membranes were incubated with primary antibodies, including rabbit anti-Bcl-2 (cat. no. ab59348; 1:1,200; Abcam, Cambridge, UK), rabbit anti- Bcl-associated X (Bax; cat. no. ab32503; 1:1,200; Abcam, Cambridge, UK), rabbit anti-GLUT-1 (cat. no. ab15309; 1:1,200; Abcam) and rabbit anti-GAPDH antibody (cat. no. ab9485; 1:1,400; Abcam) at 4°C overnight. They were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. MBS435036; 1:1,000; MyBioSource, San Diego, CA, USA) for 2.5 h at room temperature. Enhanced Chemiluminescence Reagent (Sigma-Aldrich, Merck KGaA) was used to develop signal, which was detected using MYECL™ Imager (Thermo Fisher Scientific, Inc.). Expression levels of Bcl-2 and GLUT-1 were normalized to GAPDH endogenous control using ImageJ v1.6 software (National Institutes of Health, Bethesda, MD, USA). This experiment was performed in triplicate.

SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used to perform all statistical analysis. Expression levels of proteins, mRNA and cell proliferation data were expressed as the mean ± standard deviation, and compared using unpaired Student's t-test (between 2 groups) or one-way analysis of variance followed by least significant difference test (among multiple groups). χ² test was used to analyze the associations between GASL1 (in prostate tissues and serum) and patients' clinicopathological data. Receiver operating characteristic (ROC) curve was performed using patients with PC as true positive subjects and healthy controls as true negative subjects, and all parameters were default [95% confidence interval (CI)]. Patients were divided into high and low expression group according to the median serum levels of GASL1 (1.64). Kaplan-Meier (KM) analysis was performed to plot survival curves, which were compared by log-rank test. P<0.05 was considered to indicate a statistically significant difference.

mRNA expression levels of lncRNA GASL1 in prostate tissues and sera of patients with PC and healthy controls were detected by RT-qPCR. As shown in Fig. 1A, the expression levels of lncRNA GASL1 in PC tissues were significantly lower compared with healthy control tissues (P<0.05). In addition, the expression levels of circulating lncRNA GASL1 in serum were also significantly lower in PC tissues compared with healthy controls (P<0.05; Fig. 1B). These data suggested that downregulation of lncRNA GASL1 may be involved in pathogenesis of PC.

Diagnostic values of lncRNA GASL1 expression in prostate tissues and sera for patients with PC were evaluated using ROC curve analysis. As demonstrated in Fig. 2A, the area under the curve (AUC) for GASL1 expression in prostate tissues used for PC diagnosis was 0.9076 with a standard error of 0.02718 and 95% CI of 0.8543–0.9608. In addition, the AUC of GASL1 expression in serum used for PC diagnosis was 0.8811 with a SE of 0.02976 and 95% CI of 0.8228–0.9359 (Fig. 2B). These results suggested that lncRNA GASL1 may serve as a potential biomarker for PC.

The 66 patients with PC were divided into high (n=33) and low (n=33) expression groups according to the median serum level of GASL1 (1.64). KM was used to plot survival curves, which were compared using log rank test. As presented in Fig. 3, the overall survival of patients with low serum level of GASL1 was significantly poorer compared with patients with high serum level of GASL1 (P=0.0035). These data suggested that serum levels of GASL1 may serve as a potential prognostic biomarker for PC.

Associations between expression levels of lncRNA GASL1 in prostate tissues and sera from patients with PC clinicopathological data were analyzed by χ² test. As presented in Tables I and II, expression levels of lncRNA GASL1 in prostate tissues and sera were not associated with patients age, lifestyle habits (including drinking and smoking) and existing of tumor distant metastasis. By contrast, expression levels of lncRNA GASL1 in prostate tissues and sera were associated with tumor size.

Significantly lower expression levels of GASL1 were observed in 22Rv1 and DU145 cell lines compared with the HPrEC cell line (P<0.05; Fig. 4A). According to the data in Tables I and II, GASL1 could hypothetically be involved in PC tumor growth. The effects of GASL1 overexpression on cell proliferation were assessed by CCK-8 assay. As demonstrated in Fig. 4B, GASL1 overexpression significantly inhibited 22Rv1 and DU145 cell proliferation compared with the Control groups at 96 h; however, GASL1 overexpression exhibited no observable effect on HPrEC cell proliferation. In addition, compared with the control and negative control groups, significant GASL1 overexpression was observed at different time points, which supports the effects of GASL1 overexpression on cancer cell proliferation (P<0.05; Fig. 4C).

Bcl-2, GLUT-1 and Bax are associated with development of various types of malignancy (8,9). In the present study, GASL1 overexpression (12 h following transfection) significantly increased Bcl-2 expression and significantly inhibited GLUT-1 expression in 22Rv1 and DU145 cells (Fig. 5A and B, respectively). However, GASL1 overexpression had no effect on Bcl-2 and GLUT-1 expression in the HPrEC cell line (Fig. 5C). GASL1 overexpression had no effect Bax expression in any of the three cell lines (Fig. 5A-C).