The data on CCL2 expression levels were collected from The Cancer Genome Atlas database (TCGA; cancergenome.nih.gov). In order to investigate the mechanism involved in the pathogenesis of glioma through the CCL2 pathway, 30 patients with glioma (average age of 43.23±4.84; 14 male and 16 female) were recruited from Huzhou Central Hospital, with complete clinical and pathological follow-up data, and were enrolled in the present study. Additionally, 20 non-neoplastic brain tissue samples were obtained from surgical procedures for epilepsy. Ethical approval for the present study was provided by the independent ethics committee, Shanghai Tongren Hospital (Shanghai, China). Informed written consent was obtained from all patients or their advisers according to the guidelines of the ethics committee.

U251 cells (5×10⁵/well) were seeded into 6-well plates containing antibiotic-free DMEM (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) the day prior to transfection. The cells were transfected with 50 nmol/l CCL2 siRNA or the negative control using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The sequence of the CCL2 siRNA was 5′-CTCGCGAGCTATAGAAGAA-3′.

A total of 48 h subsequently, the transfected cells were harvested and processed for proliferation, cell cycle distribution, cellular apoptosis, western blot and RT-qPCR assays.

The glioma cell line U251, purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), was cultured in DMEM (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin combination (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37°C in an atmosphere containing 5% CO₂. Cultured cells were inoculated in three 96-well plates (5×10³ cells/well) and incubated at 37°C for 12 h. A plate was designated to be the control group, and the other two plates were transfected with empty plasmid or CCL2 siRNA as the negative control and interference groups, respectively. CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and DMEM were mixed at the ratio 1:10 and added to the plates (100 µl/well) at 0, 24, 48 and 72 h post-transfection, and the plates were incubated at 37°C in 5% CO₂ for 1 h. The cell viability was determined by the optical density at 450 nm, measured using a spectrophotometer.

CCL2 siRNA-transfected U251 cells were digested following culturing for 48 h using 0.25% trypsin, followed by suspension in PBS. The cells were fixed with −20°C pre-cooling in ethanol and RNA removed with 1 mg/ml RNase A. The samples were stained with propidium iodide (PI) for 10 min at room temperature and the cell cycle distribution was measured using flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at 488 nm and analyzed using FlowJo version 7.6.1 (FlowJo LLC, Ashland, OR, USA). DNA content was used to determine the proportion of cells in each stage of cell cycle.

In addition, the effect of CCL2 siRNA on the cellular apoptosis of U251 cells was evaluated using flow cytometry. CCL2 siRNA-transfected cells were digested using trypsin and re-suspended in PBS following 24 h of culturing. Cells were centrifuged at 1,000 × g for 5 min at room temperature. The supernatant was discarded and the cells were incubated with the Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences) for 10 min at room temperature in the dark. Cellular apoptosis rate was measured and the data was obtained using FlowJo version 7.6.1 (FlowJo LLC) at a wavelength of 488 nm.

Control and CCL2 siRNA-transfected U251 cells were washed with PBS. Besides, cultured U251 cells were pre-treated with 5 µM CCR2 inhibitor RS-102895 (MCE, USA) at room temperature. Cells were fully lysed in radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd.) for 10 min and centrifuged at 12,000 × g for a further 10 min at 4°C. A total of 35 µg supernatant was subjected to 15% SDS-PAGE following quantification using bicinchoninic acid protein assay reagent (Sangon Biotech Co., Ltd., Shanghai, China). A nitrocellulose filter membrane (Merck KGaA, Darmstadt, Germany) was used to transfer the blots following soaking in transfer buffer (48 mM Tris, 39 mM glycine, 0.04% SDS and 20% methyl alcohol) for 10 min. The membrane was blocked with 5% skimmed milk (BD Biosciences) at room temperature for 1 h. Anti-caspase-3 (Ab32351; 1:5,000; Abcam Cambridge, UK), caspase-7 (Ab32522, 1:1,000; Abcam), vascular endothelial growth factor (VEGF) A (Ab115961; 1:1,000; Abcam), VEGFB (Ab185696; 1:500; Abcam), VEGF (Ab46154, 1:1,000; Abcam), CXCL1 (Santa, Sc-130316, 1:200), CXCL2 (Abcam, Ab139115, 1:100), CXCR2 (Abcam, Ab65968, 1:200; Abcam), TNFRSF10C (Sc-26462, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p38 (9212; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-p38 (9211; 1:1,000; Cell Signaling Technology, Inc.), extracellular signal-related kinase (ERK)1/2 (4695; 1:1,000; Cell Signaling Technology, Inc.), p-ERK1/2 (4376; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (5174; 1:1,500; Cell Signaling Technology, Inc.) were diluted and added to blocking buffer (Merck KGaA), followed by incubation at 4°C for 12 h. The membrane was subsequently incubated with Goat anti-rabbit (A0208; 1:1,000; Beyotime Institute of Biotechnology, Haimen, China) and anti-mouse (A0216; 1:1,000; Beyotime Institute of Biotechnology) secondary antibodies tagged with horseradish peroxidase at 37°C for 1 h. The image was developed using enhanced chemiluminescence reagents (Merck KGaA) and Tanon-5200 (Tanon Science and Technology Co., Ltd., Shanghai, China).

The gene expression level of CCL2 in glioma cell lines T98G (Thermo Fisher Scientific, Inc.), U251, and SHG44 (Type Culture Collection of the Chinese Academy of Sciences), was measured using RT-qPCR. TRIzol reagent (Gibco; Thermo Fisher Scientific, Inc.) was used to extract and quantify total mRNA from U251 cells. RT was performed to synthesize cDNA using the Thermoscript RT-PCR System (Thermo Fisher Scientific, Inc.) at a total volume of 25 µl (12 µl RNA-primer mix, 5 µl 5X RT reaction buffer, 1 µl 25 mM dNTPs, 1 µl 25 U/µl RNase inhibitor, 1 µl 200 U/µl M-MLV Rtase, 1 µl Oligo(dt)18 and 4 µl DNase-free ddH₂O).

A total PCR mix (Thermo Fisher Scientific, Inc.) of 25 µl (12.5 µl SYBR-Green mix, 0.5 µl forward primer, 0.5 µl reverse primer, 9.5 µl ddH₂O and 2 µl cDNA template) was amplified via 40 cycles of denaturing at 95°C for 15 sec, annealing at 60°C for 30 sec and elongation at 60°C for 45 sec. Amplification kinetic curves were obtained and the data was analyzed using ABI Prism 7300 SDS built-in software (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used in the real-time PCR analysis are listed in Table I.

Data are expressed as the mean ± standard deviation and were analyzed using t-tests and analysis of variance. GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to perform all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

According to the data on CCL2 expression level obtained from TCGA, glioma tissue exhibited an increase in CCL2 expression compared with healthy tissue (Fig. 1A). RT-qPCR analysis was also performed to detect the gene expression level of CCL2 in glioma tissue and healthy tissue. Similarly, compared with healthy samples, CCL2 expression level was significantly increased in the glioma tissue samples (Fig. 1B). Due to the significant alteration of CCL2 expression between glioma tissue and normal tissue, three glioma cell lines, T98G, U251 and SHG44, were used to detect the expression level of CCL2 using western blot analysis. As presented in Fig. 1C, CCL2 exhibited increased expression levels in U251 cells compared with the other cell lines. Therefore, the glioma cell line U251 was used for further investigation of the possible mechanism underlying siRNA interference in glioma.

The glioma cell line U251 was transfected with CCL2 siRNA and the results are presented in Fig. 2. The expression level of CCL2 was significantly decreased in U251 cells transfected with CCL2 siRNA compared with the control group, demonstrating the interference ability of CCL2 siRNA in glioma cell lines. Additionally, the data presented in Fig. 2D and E demonstrated that the proliferation of U251 cells transfected with CCL2 siRNA decreased in a time-dependent manner compared with the control group, measured using a CCK8 assay. Although a significant difference was observed between the negative control and control groups at 48 and 72 h post-transfection, it was hypothesized that this was due to over-proliferation and not the effect of the empty plasmid.

The cell cycle distribution of the glioma cell line U251 was detected 48 h following transfection with CCL2 siRNA using flow cytometry. The cell cycle of U251 cells was observed to be arrested at the S phase. As presented in Fig. 3A, the percentage of G0-G1 cells in the siRNA-treated U251 group decreased significantly from 64.52±1.47 to 42.16±1.80% (n=3; P<0.001) compared with the control group, while the percentage of cells in the S phase increased from 15.59±1.65 to 36.36±1.71% (n=3; P<0.001), compared with the control group. The significant increase in the percentages of cells in S phase indicated that the cell cycle of U251 cells was arrested at S phase due to the treatment with CCL2 siRNA.

The effect of CCL2 siRNA on the cellular apoptosis of the glioma cell line U251 was evaluated using flow cytometry, following treatment with CCL2 siRNA for 48 h. As presented in Fig. 3B, the cellular apoptosis induced by CCL2 siRNA in U251 cells was detected visually using Annexin V FITC/PI double staining. The percentages of early apoptotic cells located in the lower right quadrant of the histograms were recorded as the apoptotic rate and are presented in Fig. 3B. The apoptotic rate of U251 cells transfected with CCL2 siRNA increased between 2.73±0.89 and 26.57±1.14% (n=3; P<0.001) compared with the control group, demonstrating that cellular apoptosis of U251 cells was induced by treatment with CCL2 siRNA. Additionally, western blot and RT-qPCR analyses were used to evaluate the alteration in cellular apoptosis-associated proteins caspase-3, caspase-7 and TNFRSF10C. The expression levels of caspase-3 and caspase-7 were upregulated in U251 cells compared with the control, while TNFRSF10C exhibited a significant decrease in expression level (Fig. 4A-C). Consistent with the results obtained in the western blot assay, the gene expression levels measured using RT-qPCR demonstrated up- and downregulation of the caspase proteins and TNFRSF10C, respectively. The results of the present study demonstrated that the increase in the expression levels of apoptosis-associated proteins caspase-3 and caspase-7 resulted in the cellular apoptosis of U251 cells.

The expression levels of CCL2-associated pathway-associated proteins, including CXCL1, CXCL2, CXCR2, VEGFA, VEGFB and VEGF, were analyzed using western blot and RT-qPCR analysis following treatment with CCL2 siRNA for 48 h. As presented in Fig. 4A and B, the protein expression of CXCL1, CXCL2 and CXCR2, which are responsible for the proliferative and invasive capabilities of tumor cells, decreased significantly compared with the control (P<0.001). Similarly, the protein expression of vascular endothelium generation-associated proteins VEGFA, VEGFB and VEGF were observed to be significantly decreased compared with the control group (P<0.001). Consistent with the results obtained using western blotting, the gene expression levels of VEGFA (P<0.01), and CXCL1, CXCL2, CXCR2, VEGFB and VEGF (all P<0.001) exhibited a decrease compared with the control (Fig. 4C). The downregulation of biological pathway-associated proteins demonstrated the inhibitory ability of CCL2 siRNA against the proliferation and invasion of the glioma cell line U251.

The glioma cell line U251 was treated with the CCR2 inhibitor RS-102895, and the phosphorylation of p38 and ERK1/2 was measured using western blotting. The inhibition of CCR2 led to the downregulation of the expression of apoptosis-associated proteins p38 and ERK1/2. As presented in Fig. 4D and E, the phosphorylation of ERK1/2 and p38 decreased significantly compared with the control in U251 cells, due to the inhibition of CCR2 (P<0.001). The inhibition of CCR2 resulted in the downregulation of p38 and ERK1/2 expression levels, leading to cellular apoptosis in U251 cells and demonstrating the important role of the CCL2/CCR2 signaling pathway in the proliferation of the glioma cell line U251.