A total of 5 NPC cell lines HONE1 (Shanghai Jining Shiye, Shanghai, China), C666-1 (Shanghai Gaining Biotechnology Co., Ltd., Shanghai, China), CNE2, CNE1 [both from Sai Bai Sheng (Shanghai) Biotechnology Co., Ltd., Shanghai, China] and SUNE-1 [Kelton Biotech (Shanghai) Co., Ltd., Shanghai, China] were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal calf serum (Gibco; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% 100 × mycillin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with 5% CO₂ at 37°C. NPC cell lines with high expression level of TRIM24 were determined by using RT-qPCR analysis. Cell viability of selected cell lines (HONE1 and CNE1) was detected at 95% using Trypan blue staining for 5 min at room temperature. Then the cells were digested and seeded into 6-well plate (5×10⁵ cells/well) prior to transfection. Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 5 µl TRIM24 siRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) or negative control siRNA (Shanghai GenePharma Co., Ltd.) into HONE1 and CNE1 cell lines. After incubation for 48 h, the transfected cells were collected and processed for cell viability, apoptosis, biochemical tests, RT-qPCR and western blot assay.

Western blot assay was used to determine the expression levels of TRIM24 and associated proteins in NPC cell lines. Cultured or transfected cells were washed with 1xPBS twice, followed by the radioimmunoprecipitation assay lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at 4°C. Following lysis, samples were centrifuged at 12,000 × g for 15 min at 4°C and the supernatant was collected. Proteins were quantified by using the bicinchoninic acid assay. Protein (15 µl) with loading buffer were heated in a boiling water bath for 10 min and then centrifuged at 12,000 × g for 10 min at room temperature. The supernatant (30 µg protein) was collected and loaded on 10% SDS-polyacrylamide gels. Following electrophoresis, proteins on the gel were transferred onto a nitrocellulose blotting membrane (EMD Millipore, Billerica, MA, USA) and incubated with 5% non-fat milk at 4°C overnight. The membrane was washed and incubated with TRIM24 (Abcam, Cambridge, UK; cat. no. Ab174287; 1:1,000), vascular endothelial growth factor (VEGF; Abcam; cat. no. Ab46154; 1:1,000), VEGF receptor 2 (VEGFR2; Affinity, Cincinnati, OH, USA; cat. no. AF6281; 1:1,000), caspase-3 (Abcam; cat. no. Ab44976; 1:500), caspase-9 (Abcam; cat. no. Ab2013; 1:1,000), janus kinase 2 (JAK2; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 3230; 1:1,000), phosphorylated (p)-JAK2 (Cell Signaling Technology, Inc.; cat. no. 8082; 1:1,000), signal transducer and activator of transcription 3 (STAT3; Abcam; cat. no. Ab76315; 1:2,000), p-STAT3 (cat. no. 9139; 1:1,500) and GAPDH (both Cell Signaling Technology, Inc.; cat. no. 5174; 1:2,000) antibodies for 2 h at room temperature. Then the membrane was washed with TBS-0.05% Tween-20 and incubated with anti-horseradish peroxidase secondary antibody (Beyotime Institute of Biotechnology, Shanghai, China; cat. nos. A0208 and A0216; 1:1,000 dilution) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection kit and an LAS-4000 mini system (Fujifilm Corporation, Kumamoto, Japan) was used for visualization. Western blotting was repeated three times. The films of western blotting were then scanned by using a Bio-Rad imaging densitometer (ModelGS-700; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the densities of the bands were semi-quantified using Quantity One software Version 4.62 (Bio-Rad Laboratories, Inc.).

PCR analysis was preformed to determine the expression level of TRIM24 in NPC tissue samples and associated proteins in NPC cell lines. The gene expression of TRIM24 in 35 tissue samples with para-carcinoma tissues were determined using RT-qPCR. Paired human NPC tissues and para-carcinoma tissues were collected from 35 patients (including 23 males and 12 females, age range: 31 to 74 years) that underwent standard surgical procedures between January 2014 and December 2014 in Department of Otolaryngology, Zhongshan Hospital (Shanghai, China) after the written informed consent of the patients was obtained. The experimental protocols were approved by the Institutional Review Committees of Zhongshan Hospital (Shanghai, China). Tissue samples were collected from patients with complete clinical and pathological follow-up data.

Total RNA was extracted from tissue samples by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription kit (Fermentas; Thermo Fisher Scientific, Inc.) was used to syntheses cDNA through a total volume of 25 µl (12 µl RNA-primer mix, 5 µl 5XRT reaction buffer, 1 µl 25 mM dNTPs, 1 µl 25 U/µl RNase inhibitor, 1 µl 200 U/µl M-MLV reverse transcriptase, 1 µl Oligo(dt)₁₈ and 4 µl DNase-free ddH₂O) and the thermocycling conditions were: 37°C for 60 min, 85°C for 5 min and 4°C for 5 min for the RT step. Then cDNA was amplified by using SYBR-Green PCR kit (Thermo Fisher Scientific, Inc.) using a total PCR system 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) with the PCR thermocycling conditions were as follows: 10 min at 95°C, and then 15 sec at 95°C and 45 sec at 60°C for 40 cycles. Amplification kinetic curves were obtained through 15 sec at 95°C, 1 min at 60°C, 15 sec at 95°C and 15 sec at 60°C. Data was collected by using ABI Prism 7300 SDS software (Applied Biosystem, Thermo Fisher Scientific, Inc.) and quantified as previously described (18). Primers used for the amplification were listed in Table I.

Cultured HONE1 and CNE1 cells were trypsinized and diluted to 2×10⁴ cells/ml. Then cells were plated into 96-well plates (2×10³ cells/well) and incubated at 37°C for 12 h, followed by the transfection with TRIM24 siRNA or negative control siRNA, respectively. Transfected cells in each well were mixed with 100 µl DMEM containing 10% CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) at 0, 24, 48 and 72 h, and then incubated in 5% CO₂ at 37°C for 1 h. The optical density (OD)_{450 nm} of cell suspension was measured using a spectrophotometer in order to evaluate the cell viability of HONE1 and CNE1 cells.

Transfected cells were formed into a single cell suspension by 0.25% trypsin and washed by 10% PBS, followed by the centrifugation at 1,000 × g for 5 min at 4°C. Supernatant was discarded and then the cells were incubated with Annexin-V fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) for 10 min at room temperature without light. Cell apoptosis rate was measured by using flow cytometry (FACS Calibur, BD Biosciences) and FlowJo Software version 7.6 (FlowJo LLC, Ashland, OR, USA).

Caspase-3 and caspase-9 concentrations were assessed using Caspase-3 and caspase-9 colorimetric assay kits (KeyGen Biotech Co., Ltd., Shanghai, China). Cultured and transfected cells were collected and washed by using PBS for twice, followed with lysis with 100 µl pre-cooled lysis buffer containing 1 µl dithiothreitol (DTT). Lysed cells were centrifuged at 12,000 × g for 1 min. Supernatant was collected and protein concentrations were determined using the Bradford method. 50 µl supernatant was mixed with 50 µl 2X Reaction Buffer containing 0.5 µl DTT and 5 µl Caspase-3 or Caspase-9 substrate, respectively. After 4 h of incubation at 37°C, the concentrations of Caspase-3 and Caspase-9 were measured at OD 400 nm.

All the experiments were repeated three times and the values are expressed as the mean ± standard deviation. Statistical comparisons were performed using one-way analysis of variance followed by the Tukey post hoc test with GraphPad Prism software (version 6.0; Graphpad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1A, the increased expression level of TRIM24 measured by RT-PCR in NPC tissues demonstrated a significant difference compared with the paracancerous tissues, indicating the overexpression of TRIM24 in NPC tissues and suggesting an association between TRIM24 and NPC. To further investigate the association between TIRM24 and NPC, NPC cell lines HONE1 and CNE1 with higher expression levels of TRIM24, as determined using western blotting, were selected for further experiments (Fig. 1B and C).

Furthermore, western blotting and qPCR were used to identify the interference effect of TRIM24 siRNA on the expression of TRIM24 in HONE1 and CNE1 cells. As presented in Fig. 1D and E, the expression level of TRIM24 declined significantly in HONE1 and CNE1 cells (P<0.05 or P<0.001). The gene expression of TRIM24 was additionally decreased, with a significant difference compared with the negative control group (Fig. 1F; P<0.001), indicating the efficient gene silencing of TRIM24 siRNA.

As presented in Fig. 2, the cell viability of HONE1 and CNE1 cells detected via CCK8 assay was decreased following treatment with TRIM24 siRNA, in a time-dependent manner. The decreased cell viability was significant compared with the control and negative control groups, indicating the inhibitory effect of TRIM24 siRNA on cell proliferation in HONE1 and CNE1 cells.

The results of the flow cytometry demonstrated cellular apoptosis in HONE1 and CNE1 cells induced by TRIM24 siRNA (Fig. 3). The percentages of early apoptotic cells presented in the lower right quadrant of the histograms indicated the induced cellular apoptosis in the TRIM24 siRNA group, suggesting an apoptosis-promoting effect of TRIM24 siRNA on HONE1 and CNE1 cells. Furthermore, the expression of the pro-apoptotic proteins caspase-3 and caspase-9, measured using biochemical analysis, exhibited an upregulation following treatment with TRIM24 siRNA, which corresponded to the results of the flow cytometry (Fig. 3B and D). Additionally, the upregulated caspase-3 and caspase-9 measured by western blotting and qPCR indicated the pro-apoptotic ability of TRIM24 siRNA on HONE1 and CNE1 cells, suggesting an important role for TRIM24 in NPC (Fig. 3E-G).

To further investigate the possible mechanisms involved in inhibited cellular apoptosis and cell proliferation due to TRIM24 siRNA, the expression levels of the cellular apoptosis- and tumorigenesis-associated proteins caspase-3, caspase-9, VEGF and VEGFR2 in HONE1 and CNE1 cells was measured by western blotting and qPCR. Compared with the control group, the expression levels of VEGF and VEGFR2 exhibited a downregulation with a significant difference in HONE1 and CNE1 cells transfected with TRIM24 siRNA (Fig. 4A and B; n=3; P<0.001). Similar results were observed with the PCR analysis (Fig. 4C). Besides, the expression levels of the pro-apoptotic proteins caspase-3 and caspase-9 exhibited an upregulation, corresponding to the results of the flow cytometry. In addition, the expression of the JAK2/STAT3 signaling pathway-associated proteins JAK2 and STAT3 was measured using western blotting and qPCR. As presented in Fig. 4A, D and E, decreased expression of JAK2 and STAT3 indicated the inhibition of the JAK2/STAT3 signaling pathway. However, further evidence is required to demonstrate the direct action of siTRIM24 on the inactivation of JAK2/STAT3 signaling pathway.