Human glioma tissues were obtained from the surgical branch of Tangdu Hospital, Fourth Military Medical University, China. Normal brain tissues, which were preserved in liquid nitrogen, were obtained from the patients who had undergone partial brain resection. The tissue samples were obtained with the written informed consent of the patients, and the process was approved by the Ethics Committee of Tangdu Hospital Institutional Review Board.

The established U251 and U87MG cell lines used in the present study were purchased from the Cell Bank of Chinese Academy of Sciences (14). The authenticity of the cancer cell lines was tested by short tandem repeat (STR) profiling. The STR result demonstrated that the U87 cell line used in the present study matched the U87MG cell from ATCC. However, the U87MG cell line from ATCC may not be the original GBM cell line from the University of Uppsala established in 1968. It has been reported that the U87MG cell line from ATCC is of CNS origin and is likely to be derived from another patient with glioma, although its source is unknown (15). Therefore, the aforementioned misidentifications of U87MG-ATCC did not affect the outcomes of the present study. All the cell lines were cultured in 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM; both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The CADM2 coding sequence (CDS) was sub-cloned into a pCDH1-overexpression vector using the restriction sites of Bamh1 and Xho1. U251 and U87 cells were transfected with CADM2-overexpression lentivirus, and stable CADM2-overexpression cell lines were established by puromycin screening at 2 µg/ml.

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Reverse transcription was performed using a Takara reverse transcription kit (Takara Bio, Inc., Otsu, Japan). The sequences of the primers used were as follows: CADM2 forward, 5′-CCTCAATGCCACCCCTCAG-3′ and reverse, 5′-TTCTCCGCCATCCTTTGTCC-3′; and β-actin forward, 5′-TCCCTGGAGAAGAGCTACG-3′ and reverse, 5′-GTAGTTTCGTGGATGCCACA-3′. The reverse transcription process was as follows: 37°C for 1 h and 85°C for 15 sec. PCR conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 20 sec, 60°C for 30 sec, 72°C for 60 sec. Gene expression in each sample was normalized to relative to that of β-actin mRNA. qPCR was performed using SYBRGreen (Takara Bio, Inc.) with the ABI PRISM 7700 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression was normalized to human β-Actin expression using the 2^−ΔΔCq method (16).

Radioimmunoprecipitation assay (RIPA) lysis buffer was used to lyse glioma tissues and different glioma cell lines, and complete protease inhibitor cocktail (Beyotime Institute of Biotechnology, Haimen, China) was supplemented with RIPA lysis buffer prior to lysis. Bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to detect protein concentration. A total of 30 µg protein samples were loaded onto the gel and separated by 12% SDS-PAGE gel. Protein was subsequently transferred to polyvinylidene difluoride membranes. Following blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4°C overnight: CADM2 (dilution, 1:4,000; cat. no. ab64873; Abcam, Cambridge, UK), β-actin (dilution, 1:4,000; cat. no. M20011; Abmart Co., Ltd., Shanghai, China), CDK2 [dilution, 1:2,000; cat. no. 2546; Cell Signaling Technology (CST)], CDK4 (dilution, 1:2,000; cat. no. 12790; CST), E-cadherin (dilution, 1:2,000; cat. no. 14472; CST), β-catenin (dilution, 1:2,000; cat. no. 8480; CST), cyclin D1 (dilution, 1:1,000; cat. no. sc-70899; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and cyclin E (dilution, 1:1,000; cat. no. sc-377100; Santa Cruz Biotechnology, Inc.). On the second day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse IgG, cat. no. ab97023 or goat anti-rabbit IgG, cat. no. ab6721; dilution, 1:1,000) at room temperature for 2 h. Next, the membranes were exposed using a Millipore enhanced chemiluminescence kit.

The proliferation rates of different U87 and U251 cells (Blank, vector and CADM2 groups) were detected using an MTT assay. A total of 1×10⁴ cells/well were suspended in 200 µl culture DMEM supplemented with 10% FBS and seeded in 96-well plates. Every 24 h after cell seeding, the culture medium of the 96-well plate was replaced with DMEM, and then incubated at 37°C for 4 h. Next, DMEM was replaced with isopyknic dimethyl sulfoxide. Following 5 shocks, each of 1 sec duration, the absorbance at 490 nm was detected by SpectraMax 190 (Molecular Devices, LLC, Sunnyvale, CA, USA) to measure the optical density (OD) value.

Wound-healing assay was performed in 6-well culture plates. In brief, 5×10⁵ cells were suspended in medium containing 10% FBS, and were then seeded into 6-well plates until monolayer cells were nearly confluent. A 200-µl sterile pipette tip was used to equably wound the monolayer cells, and the cells were washed with PBS twice to remove the floating cells. Following incubation in DMEM supplemented with 1% FBS for 24 h, images of the wounded monolayers of blank, vector and CADM2 glioma cell groups were captured under a phase-contrast microscope (magnification, ×20; Olympus Corporation, Tokyo, Japan).

QCM 24-Well Cell Invasion assay kit (EMD Millipore, Billerica, MA, USA) was used for the Transwell invasion assay processed by a Transwell filter coated with Matrigel. Cells were seeded onto the top side of the Transwell filter on the top chamber. DMEM supplemented with 10% FBS was added to the bottom chamber. Cells (0.5×10⁵) were suspended in 250 µl serum-free DMEM. After 72 h incubation, the residual cells were removed from the top chamber with a cotton swab, and the invaded cells were dislodged from the lower membrane surface using detachment solution. The detachment cells were stained with Hoechst 33342 at 37°C for 30 min, and then images of the detached cells were directly captured and the number of cells was counted under a fluorescence microscope (magnification, ×20). Cells were counted in at least 5 randomly selected fields of view for each group. The number of invaded cells per field was calculated and data are presented as the mean ± standard error of the mean.

The stably transfected negative control (NC) and CADM2 lentivirus U87 cells (2×10⁶ of each) were suspended in PBS and then implanted subcutaneously into 10 male Balb/c nude mice aged 4 weeks. The mice were purchased from Vital River Laboratories Co., Ltd. (Beijing, China). The length (a) and width (b) of NC and CADM2 mice tumors were monitored every 2 days. After 3 days of experiments, the nude mice were sacrificed and the weights of the animals ranged between 24.3 and 25.8 g. Tumor volume (V) was calculated using the formula V = ab²/2. Animal experiments in the present study had been approved by the Ethics Committee of Tangdu Hospital Institutional Review Board.

Data are presented as the mean ± standard deviation of at least three independent experiments. Significance between groups was analyzed by one-way analysis of variance, followed by a Newman-Keuls comparison test using GraphPad Prism version 5.03 (GraphPad Software, Inc., La Jolla, CA, USA). *P<0.05, **P<0.01 and ***P<0.001 were considered to indicate statistically significant differences.

To investigate the candidate role of CADM2 in human glioma, the data of CADM2 in the Oncomine database was initially referred to. It was demonstrated that the mRNA expression level of CADM2 was markedly decreased in tumor tissues (98 samples) compared with normal brain tissues (3 samples; Fig. 1A). Tissue microarrays were performed on 60 glioma tissues (grade I, II, III and IV), 10 cancer adjacent non-cancerous brain tissues and 10 normal brain tissues to compare the protein expression level of CADM2 in normal tissues and different grades of the malignancy (Fig. 1A). Tissue microarray results demonstrated that CADM2 expression was much lower in higher grade tissues, particularly grade II, III and IV glioma samples, than in the grade I samples (Fig. 1B). The CADM2 expression levels in different grades of glioma tissues and normal tissues were studied by immunohistochemistry. Representative images of these tissues are presented in Fig. 1C. Next, survival analysis was performed to study the association between CADM2 expression and survival times in patients with glioma. The Kaplan-Meier survival curves presented in Fig. 1D revealed that glioma patients with high CADM2 expression had a longer overall survival time than those with low CADM2 expression (P<0.05). Western blot analysis also verified the lower expression level of CADM2 in 4 GBM tissues, compared with the 4 corresponding adjacent non-cancerous brain tissues (Fig. 2A and B). The results also demonstrated that CADM2 exhibited a much lower expression in glioma tumor tissues. In addition, CADM2 protein expression in the U87 and U251 cells was almost undetectable, while in normal brain cells it was particularly high (Fig. 2C), and the mRNA expression level of CADM2 in the GBM U87 and U251 cell lines was ~60% lower than in the normal brain (Fig. 2D). Taken together, these data indicated that the low expression level of CADM2 at the mRNA and protein expression levels was observed in human glioma.

To verify the association between CADM2 expression and GBM development, all 98 patients were stratified into CADM2-low and CADM2-high groups using the median value of CADM2 expression. Of the 98 patients with gliomas, 80 (81.63%) belonged to the CADM2-low and 18 (18.37%) to the CADM2-high groups, respectively. As demonstrated in Table I, low CADM2 expression occurred more frequently in glioma patients with advanced WHO grade (II–IV). However, no statistically significant differences were observed between CADM2 expression and other demographic characteristics, including patient age and sex (P>0.05; Table I).

To investigate the biological function of CADM2 on glioma tumorigenesis, CADM2 was overexpressed in U87 and U251 cell lines, respectively (Fig. 3A and C). The effect of CADM2 on glioma cell proliferation was measured using an MTT cell proliferation assay. The results demonstrated that the overexpression of CADM2 significantly inhibited the proliferation of U87 and U251 compared with the blank group and the vector group (Fig. 3B and D). Nude mice xenograft tumors were created to verify the effect of CADM2 on glioma proliferation in vivo. The CADM2-overexpressing U87MG cells and the control U87MG cells were suspended in PBS and implanted into two sides of the mice. After 30 days, the tumor volume results demonstrated that CADM2 significantly inhibited U87MG xenograft tumor growth in vivo. The mean volumes of CADM2-U87 tumors cells were almost half the size of that of the control U87MG cells (n=10 animals per group, Fig. 3E and F). Ki-67 staining revealed that there were fewer Ki-67-positive cells in CADM2-U87MG tumors than in U87MG control tumors (Fig. 3G and H). Therefore, these results demonstrated that the CADM2 overexpression significantly inhibited glioma proliferation in vitro and in vivo.

Next, a wound-healing assay and a Transwell assay were performed to study the role of CADM2 on glioma cell migration and invasion. In the wound-healing assay, the healing rate was calculated by the healing distance divided by the initial distance in order to demonstrate the cell migration ability. The results indicated that CADM2 inhibited the healing rate of glioma cells, and the inhibition rate of U87 and U251 cells was 57.6% (Fig. 4A and B) and 52.5% (Fig. 4C and D), respectively. The Transwell experiment was processed to examine the migratory capacity of glioma cells, and at least three wells were used for each experiment. The results demonstrated that the overexpression of CADM2 markedly attenuated the invasive properties of U87 and U251 cells; the number of invaded cells per field declined from 62 to 38/field for U87 cells (Fig. 4E and G) and from 52 to 25/field for U251 cells (Fig. 4F and H), respectively. The results indicated that CADM2 could inhibit the migratory and invasive abilities of glioma cells.

CADMs were reported as key regulators in G1/S transition. The results of the present study demonstrated that CADM2 overexpression significantly decreased the protein expression levels of cyclin D1, CDK4, cyclin E1 and CDK2, compared with the blank group (Fig. 5A and B). The EMT is a crucial process for the invasion of cancer cells (17,18). Next, the expression of two EMT markers, E-cadherin and β-catenin, was detected by western blot analysis. The results demonstrated that CADM2 significantly downregulated the protein expression level of β-catenin and markedly increased the expression level of E-cadherin in U87 cells (Fig. 5A) and U251 cells (Fig. 5B). These data suggested that CADM2 may inhibit glioma cell proliferation by regulating cell cycle related genes and inhibit glioma cell invasion via regulating the glioma cell EMT process.