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Introduction

Nasopharyngeal carcinoma is a radiotherapy-sensitive tumor, but with the extension of radiotherapy, the percentage of hypoxic cells gradually increased (up to 10 to 50%), and the increased percentage of hypoxic cells can lead to the failure of solid tumor chemotherapy, the recurrence and metastasis of tumors (1). Tumor cell hypoxia on the one hand can reduce the production of oxygen free radicals, resulting in reduction in radiotherapy-induced DNA breakage, on the other hand can increase the release of hypoxia-inducible factor-1 (HIF-1), increase the expression of vascular endothelial growth factor (VEGF), and inhibit tumor cell apoptosis (2). Therefore, the use of hypoxic cytotoxic drugs combined with radiotherapy and chemotherapy can improve the efficacy of cancer treatment. Toll-like receptor (TLR), as an important component of innate immunity, is related to the pathogenesis of nasopharyngeal carcinoma, breast cancer, pancreatic cancer, basal cell carcinoma and other malignant tumors (3,4). TLR-9/myeloid differentiation factor 88 (MyD88) signaling pathway can activate inflammatory and immune responses (5), mediate cell proliferation and apoptosis regulated by nuclear transcription factor (NF-κB) (6), stimulate the secretion of matrix metalloproteinases and integrins, and induce tumor cell invasion and metastasis (7). Based on this, we investigated the correlation between the expression of Toll-like receptor-9 (TLR-9) and cell proliferation and apoptosis in hypoxic nasopharyngeal carcinoma cells.

Materials and methods

Experimental materials

Human nasopharyngeal carcinoma cell line HNE-1 (EBV positive) and CNE-1 (EBV negative) were purchased from Sangon (Shanghai, China). Occurrence of nasopharyngeal carcinoma is closely related to EB virus infection, so both EBV-positive and -negative cell lines were used. Cells were cultured with RPMI-1640 cell culture medium (Beyotime Biotechnology, Jiangsu, China) containing 10% fetal bovine serum, 100 U/ml penicillin and 40 U/ml gentamicin in an incubator (37°C, 5% CO2). Cell recovery and subculture were performed using the same method. Cells were collected at logarithmic growth phase for following experiments.

Experimental methods

Cells were divided into normal control group, hypoxia group and hyperoxia group. Hypoxic conditions were 5% CO2 and 0.01% partial pressure of oxygen, hyperoxia conditions were 5% CO2 and 10% partial pressure of oxygen. After culture for 6, 12 and 24 h, cells in hypoxia group and hyperoxia group were cultured in normoxic condition for another 4 h. RT-PCR and western blot analysis were used to detect the expression of TLR-9 mRNA and protein at 6, 12 and 24 h after the beginning of cell culture. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the cell proliferation rate and flow cytometry was used to detect the cell apoptosis rate.

Detection method

Total RNA was extracted by 1 ml of TRIzol reagent (Beyotime Biotechnology, Jiangsu, China). The purity and concentration of RNA samples were checked by 1.5% agarose gel electrophoresis and UV spectrophotometer (HyClone, Logan, UT, USA). cDNA was synthesized using 2 µg total RNA and reverse transcription kit (Sigma, St. Louis, MO, USA). Primers were designed by Sangon (Shanghai, China) accroding to the gene sequences downloaded from GenBank. The following primers were used: TLR-9 forward, 5′-GGTTTCATCCAGGATCGAGCAGG-3′ and reverse, 5′-ACAAAGATGGTCACGGTCTGCC-3′; endogenous control GAPDH forward, 5′-CGCGAGAAGATGACCCAGAT-3′ and reverse, 5′-GCACTGTGTTGGCGTACAGG-3′. The PCR reaction system was: 2 µl of cDNA + 3 µl of each primer + 0.5 µl of Taq polymerase (Sigma) + 1 µl of dNTPs + 2 µl of 10X buffer, water was added to make a final volume of 20 µl. Reaction conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec, and 72°C for 10 min. PCR product (6 ml) was subjected to 2% agarose gel electrophoresis, and results were checked and pictures were taken using a gel imaging system. The grey scale values were analyzed.

RIPA lysate (Beyotime Biotechnology) was added to extract total protein, and BCA quantitative kit (Beijing Zhongshan Golden Bridge Biology Co., Ltd., Beijing, China) was used to quantify protein, and β-actin was used as endogenous control for normalization. Thirty micrograms of protein from each sample was subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, followed by transmembrane to PVDF membrane. Membrane was then incubated with mouse anti-human TLR-9 monoclonal primary antibody (dilution, 1:500; cat. no. 12-9099-82; Thermo Fisher Scientific Inc., MA, USA) or mouse anti-human β-actin primary antibody (dilution, 1:2,000; Sigma-Aldrich, Inc., MA USA) overnight at 4°C. After washing, membranes were incubated with goat anti-mouse polyclonal secondary antibody (1:500; Sigma-Aldrich, Inc.) at room temperature for 4 h. After washing with PBS, color development was performed with ECL. Results were scanned and recorded. Lab Works 4.5 gel imaging software (Invitrogen, Carlsbad, CA, USA) was used for semi-quantitative analysis.

Cells were resuspended to make a concentration of 2×106/ml and transferred to a 96-well plate with 100 µl for each well. After incubation for 6, 12 and 24 h, 10 µl of 5 mg/ml MTT (Bio-Rad, Hercules, CA, USA) was added to each well, followed by incubation for another 4 h. Culture medium was discarded and 150 µl of dimethyl sulfoxide (DMSO; Bio-Rad) was added to each well and shaken for 10 min. Optical density (OD) at A490 nm was measured by a microplate reader (Bio-Rad). OD values were measured 3 times and the average value was calculated. Cell proliferation rate = sample/control OD value ×100%.

Cells were centrifuged at 1,500 × g for 10 min to remove the supernatant. After washing with PBS, cells were collected. Cells were mixed with 100 µl of binding buffer and 10 µl of FITC-labeled Annexin V (20 µg/ml) (both from Beyotime Biotechnology), followed by incubation at room temperature for 30 min. After that, 5 µl of propidium iodide (PI, 50 µg/ml; Beyotime Biotechnology) was added and incubated at room temperature for 5 min. After that, 400 µl of binding buffer was added and detection was performed within 1 h. Cell solution without Annexin V-FITC and PI was used as negative control. FACSCalibur flow cytometry (BD Biosciences, Lake Franklin, NJ, USA) was used here.

Statistical analysis. Statistical analysis was performed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Measurement data were expressed as mean ± standard deviation, comparisons among multiple groups were performed using single factor ANOVA analysis, comparisons between two groups were performed using LSD-t method, and comparisons between different time-points were performed using analysis of variance of repeated measure data. P<0.05 was considered to be statistically significant.

Results

Comparison of expression level of TLR-9 mRNA between groups. Expression level of TLR-9 mRNA in hypoxia group reached the peak at 12 h after the beginning of cell culture, and was significantly higher than that of hyperoxia group at all time-points, control group was the lowest, difference between groups were all statistically significant (P<0.05). No significant changes in expression level of TLR-9 mRNA were found in control group and hyperoxia group between different time-points (P>0.05) (Fig. 1).

Figure 1. Expression levels of Toll-like receptor-9 (TLR-9) mRNA in each group at different time-points detected by RT-PCR. #Expression level of TLR-9 mRNA in hyperoxia group was significantly higher than that in control group at each time-point; ##expression of TLR-9 mRNA in hypoxia group was significantly higher than that in hyperoxia group at each time-point; *expression level of TLR-9 mRNA in hypoxia group reached the peak.

Comparison of expression level of TLR-9 protein between groups. Expression level of TLR-9 protein in hypoxia group reached the peak at 12 h after the beginning of cell culture, and was significantly higher than that of hyperoxia group at all time-points, control group was the lowest, difference between groups were all statistically significant (P<0.05). No significant changes in expression level of TLR-9 protein were found in control group and hyperoxia group between different time-points (P>0.05) (Fig. 2).

Figure 2. Expression levels of Toll-like receptor-9 (TLR-9) protein in each group at different time-points detected by western blot analysis. #Expression level of TLR-9 proetin in hyperoxia group was significantly higher than that in control group at each time-point; ##expression of TLR-9 proten in hypoxia group was significantly higher than that in hyperoxia group at each time-point; *expression level of TLR-9 protein in hypoxia group reached the peak.

Comparison of cell proliferation rate among groups. Compared with other two groups, cell proliferation rate was gradually decreased in hypoxia group, significant differences were found between hypoxia group, and control group and hyperoxia group (P<0.05), no significant differences were found between control group and hyperoxia group (P>0.05) (Fig. 3).

Figure 3. Cell proliferation rate in each group detected by MTT assay at different time-points. #Cell proliferation rate of hypoxia group was significantly higher than that of the other two groups at each time-point; *cell proliferation rate of hypoxia group at 12 h after the beginning of culture was significantly higher than that at 6 h after the beginning of culture; **cell proliferation rate of hypoxia group at 24 h after the beginning of culture was significantly higher than that at 12 h after the beginning of culture.

Comparison of cell apoptotic rate among groups. Compared with other two groups, cell apoptotic rate was gradually decreased in hypoxia group, significant differences were found between hypoxia group, and control group and hyperoxia group (P<0.05), no significant differences were found between control group and hyperoxia group (P>0.05) (Fig. 4).

Figure 4. Cell apoptotic rate in each group detected by flow cytometry at different time-points. #Cell apoptotic rate of hypoxia group was significantly lower than that of the other two groups at each time-point; *cell apoptotic rate of hypoxia group at 12 h after the beginning of culture was significantly lower than that at 6 h after the beginning of culture; **cell apoptotic rate of hypoxia group at 24 h after the beginning of culture was significantly lower than that at 12 h after the beginning of culture.

Discussion

Studies have shown that solid tumors can overexpress HIF-1α in hypoxic microenvironment and regulate the expression of a series of genes that are compatible with hypoxia to maintain metabolic stability and promote tumor growth and metastasis. HIF-1α can exert anti-apoptotic and pro-apoptotic effects simultaneously, HIF-1α can increase the anaerobic metabolism and glucose extraction, and downregulate the expression of apoptotic genes to play an anti-apoptotic role (8); at the same time, HIF-1α can increase the level of p53 protein by inhibiting its degradation to play a pro-apoptotic role (9). Therefore, how to regulate HIF-1α to increase tumor cell apoptosis is an important task for studies on treatment of tumors.

This study showed that expression levels of TLR-9 mRNA and protein in hypoxia group reached the peak at 12 h after the beginning of cell culture, and were significantly higher than those of hyperoxia group at all time-points, expression levels of TLR-9 mRNA and protein of control group were the lowest, differences between groups were all statistically significant (P<0.05). No significant changes in expression levels of TLR-9 mRNA and protein were found in control group and hyperoxia group between different time-points; compared with other two groups, cell proliferation rate was gradually decreased and apoptotic rate was gradually decreased in hypoxia group, significant differences were found between hypoxia group, and control group and hyperoxia group, but no significant differences were found between control group and hyperoxia group, indicating that hypoxic nasopharyngeal carcinoma cells can highly express TLR-9 to regulate cell proliferation and apoptosis, which may be an important mechanism of tumorigenesis and a potential target for intervention therapy.

Clinical study of nasopharyngeal carcinoma showed that (10,11), expression level of TLR-9 was significantly higher in tumor cells than in adjacent normal tissue and the tissue from healthy volunteers, and was closely related to tumor clinical stage, pathological grade and the efficacy of radiotherapy and chemotherapy. TLR-9-1486T/CCC genotype can reduce the sensitivity of nasopharyngeal carcinoma patients to radiotherapy, promote tumor proliferation, migration and recurrence, and upregulate the expression of VEGF and other cytokines (12,13). TLR-9/MyD88 signaling pathway plays an important role in hypoxia and inflammatory responses, the expression of HIF-1α and VEGF (14), differentiation and activation of immune cells (15), and release of inflammatory factors such as IL-6 and TNF-α (16). In addition, TLR-9/MyD88 signaling pathway can mediate the transcription of NF-κB, whereas upstream promoter region of NF-κB contains HIF-1α binding site, which can affect the transcription and expression of HIF-1α, so NF-κB can participate in HIF-1α-regulated tumor cell proliferation and apoptosis and other biological activities (8,17).

The innovation of this study is that the hypoxic environment can induce high expression level of TLR-9 in nasopharyngeal carcinoma cells, which may affect the cell proliferation and apoptosis, and this may be an important mechanism of tumorigenesis and potential target of intervention therapy. Further studies may focus on the effects of TLR-9 gene invention on tumor development, so as to provide reference for clinical treatment.

References