A total of 69 fresh GC tissue specimens, together with matched adjacent non-tumorous tissue specimens, were collected from patients who underwent surgery at the Xiaogang Hospital (Ningbo, China) between May 2011 and June 2012. Written consent to use the samples for research was obtained from all patients prior to surgery. None of the patients received preoperative therapy prior to surgical resection. The histologic diagnosis of tumors was performed by at least two experienced pathologists based on the World Health Organization criteria (14). All samples were immediately frozen following resection in liquid nitrogen and stored at −80°C until RNA extraction. The study was approved by the Institutional Review Board and Human Ethics Committee of Xiaogang Hospital.

Human umbilical vein endothelial cells (HUVECs), 4 human GC cell lines (MKN-1, SGC7901, BGC823 and MGC803) and the nonmalignant epithelial gastric cell line, GES-1 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin, 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

lncRNA-CASC2 cDNA was constructed by cloning the cDNA sequence of CASC2 into the pcDNA3.1 expression vector (Invitrogen; Thermo Fisher Scientific, Inc.) by Shanghai Furui Science & Technology Co., Ltd. (Shanghai, China). For the transfection of the pcDNA-CASC2, GC cells were transfected with control vector or pcDNA-CASC2 constructs at a final concentration of 1 µg/µl using Lipofectamine^® 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following transfection for 48 h, cells were harvested for RT-qPCR analysis and the subsequent experiments.

The capability of cellular proliferation was assessed via Cell Counting Kit-8 assay (Dojingdo Molecular Technologies, Inc., Kumamoto, Japan). Approximately 3×10³ cells transfected with control vector or CASC2 were seeded into 96-well culture plates. CCK-8 solution (10 µl) was added and incubated at 37°C for 2 h and maintained for 24, 48, 72 and 96 h. Finally, the absorbance at a wavelength of 450 nm was measured using a spectrophotometer reader (Elx800; BioTek Instruments, Inc., Winooski, VT, USA). All experiments were performed as 5 replicates and were repeated 3 times independently.

Total RNA from the frozen samples and cell lines was extracted using Invitrogen TRIzol reagent; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA from all samples was synthesized from 500 ng total RNA using a PrimeScript^® 1st strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI 9700 DNA thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. qRT-PCR analyses were performed using an SYBR Premix EX Taq™ II kit (Biotechnology Co., Ltd.) on the ABI Prism 7900. The thermal cycling conditions involved initial denaturation for 10 sec at 94°C, followed by 40 cycles of 5 sec at 94°C and 30 sec at 60°C. The PCR primers for CASC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: Forward, 5′-GCTGATCAGAGCACATTGGA-3′ and reverse 5′-ATAAAGGTGGCCACAACTGC-3′ for CASC2; forward, 5′-ACCCACTCCTCCACCTTTGAC-3′ and reverse, 5′-TGTTGCTGTAGCCAAATTCGTT-3′ for GAPDH. The expression level of CASC2 was calculated using the 2^−ΔΔCq method and normalized to the respective GAPDH expression level (15). The specificity of each PCR reaction was confirmed by melting curve analyses.

For the apoptosis assays, cells were harvested with trypsin enzyme and washed with phosphate-buffered saline 48 h after transfection. Annexin-V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) were added to stain the cells according to the manufacturer's protocols. Finally, Apoptotic cells were measured with a Beckman Coulter FC500 (Beckman Coulter, Inc., Brea, CA, USA) and analyzed using FlowJo version 10.07 (FlowJo LLC, Ashland, OR, USA).

GC cells were transfected with control vector or pcDNA-CASC2. After transfection (48 h), the supernatants were collected and centrifuged at 14,000 × g for 30 min at 4°C to discard cell debris. The supernatant was harvested for further investigations. A tube formation assay was performed as previously described (16). HUVECs were cultured in conditioned medium from the supernatant of the GC cells at a density of 20,000 cells per well in a 96-well plate precoated with 150 µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The HUVECs were cultured at 37°C in a 5% CO₂ atmosphere for 6 h. The formation of HUVEC tubular structures was calculated and imaged under a light microscope (Olympus Corp., Tokyo, Japan) at a magnification of ×100.

To assess the cell motility ability in vitro, transwell chambers with 8-µm pore polycarbonate membranes (Corning Incorporated, Corning, NY, USA) were used. Following transfection (24 h), cells were collected and re-suspended in 100 µl serum-free medium, and seeded on the Matrigel-coated upper chamber. The lower chamber contained complete medium as a chemoattractant. Following 48 h incubation at 37°C, the cells on the lower side of the membrane were fixed and stained with 0.5% crystal violet solution (Beyotime Institute of Biotechnology, Haimen, China). Five fields of vision were randomly selected and cell numbers were counted under a light microscope (Olympus Corp.).

All data are expressed as the mean ± standard deviation of at least three independent experiments. The difference between two groups was estimated using Student's t-test. The χ² test was used to assess the association between the expression of CASC2 and clinicopathological features. The OS rates were analyzed using the Kaplan-Meier method, with the log-rank test applied for comparison. All statistical analyses were performed using SPSS version 11.5 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Previous studies indicated that the expression level of CASC2 was decreased in GC tissues and colorectal cancer (13,17). The expression level of lncRNA CASC2 was initially confirmed in GC tissue samples and the results indicated that the relative expression of CASC2 in GC tissue was significantly decreased compared with the non-tumor samples (Fig. 1A). The CASC2 expression level was markedly decreased in the GC cell lines compared with the normal gastric epithelial cell line, GES-1 (Fig. 1B). As SGC7901 and MGC803 exhibited relatively low endogenous CASC2 expression levels among all of the tested cell lines, these two cell lines were selected for the subsequent experiments.

In order to establish whether the expression level of CASC2 in GC tissues exerted an effect on patient clinicopathological features, the correlation between CASC2 expression level and clinicopathological features were further assessed. The 69 primary GC patients were divided into two groups based on the mean value of CASC2 in tumor tissue samples: A high-CASC2 group (n=34; CASC2 expression ratio >median) and a low-CASC2 group (n=35; CASC2 expression ratio ≤median). The associations between CASC2 expression level and the clinicopathological features are summarized in Table I. Notably, low CASC2 expression levels in GC samples were significantly correlated with vessel invasion (P=0.016), advanced TNM stage (P=0.0063) and metastasis (P=0.034). However, no significant difference between CASC2 expression level and sex, age, tumor size, cell differentiation, or tumor site was identified. To determine the association between CASC2 expression level and the prognosis of GC patients, OS curves were plotted according to the CASC2 expression level and analyzed using the Kaplan-Meier method and log-rank test. The high-CASC2 expression group had significantly shorter OS than the low-CASC2 expression group (Fig. 2). These results indicate that CASC2 may present as a useful marker of the prognosis or progression of GC.

To assess the biological role of CASC2 in GC, the effect of CASC2 on GC cell lines was evaluated. CASC2 was overexpressed by transfecting the pcDNA3.1-CASC2 vector into the SGC7901 and MGC803 cell lines, which contained the lowest expression level of CASC2. RT-qPCR analysis was performed 48 h post-transfection and the data revealed that ectopic overexpression of CASC2 significantly increased the expression level of CASC2 in SGC7901 and MGC803 cell lines (Fig. 3A). To further identify the function of CASC2 on cell growth, cell proliferation and colony formation assays were performed. As shown in Fig. 3B, cell proliferation was significantly inhibited when CASC2 was overexpressed in the SGC7901 and MGC803 cells. Furthermore, compared with the control vector, CASC2-overexpressing SGC7901 and MGC803 cells markedly induced cell apoptosis. These data indicated that CASC2 acted as a tumor suppressor by suppressing proliferation and inducing GC cells.

As a significant difference between the expression level of lncRNA CASC2, and vessel invasion and metastasis was identified, whether CASC2 is involved in GC cell invasion was then investigated. The invasion assay indicated that overexpression of lncRNA CASC2 significantly inhibited cell invasion in SGC7901 and MGC803 cells (Fig. 4A). Furthermore, the results demonstrated that formation of capillary-like structures of HUVECs treated with CASC2-conditioned medium were significantly decreased (Fig. 4B). These results revealed that CASC2 acted as a tumor suppressor via inhibiting cell invasion and angiogenesis.