The MGC-803 human gastric cancer cell line was obtained from the Type Culture Collection Center of Chinese Academy of Science (Shanghai, China) and the sh-CD14 stable CD14-knockdown MGC-803 cell subline was constructed in our previous study (9). The cells were stored in the laboratory and cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/l streptomycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in a humidified incubator with 5% CO₂ at 37°C. The cells were passaged using 0.05% trypsin-EDTA (Beyotime Institute of Biotechnology, Haimen, China) every 2–3 days and the cells at the logarithmic phase were used in the present study. The cells were treated with 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) for 4 h and harvested for in vitro study, and treated with 10 μg/ml LPS for 16 h for the tumor xenograft assay. Cells were divided into the following groups: Wild-type (WT) cells (MGC-803 cells without any interference), sh-CD14 cells (MGC-803 cells subjected to stable CD14-knockdown), WT + LPS cells (MGC-803 cells treated with 1 μg/ml LPS for 4 h or 10 μg/ml LPS for 16 h) and the LPS + sh-CD14 group (MGC-803 cells subjected to stable CD14-knockdown treated with 1 μg/ml LPS for 4 h or 10 μg/ml LPS for 16 h). The present study was approved by the Institutional Review Board of the People's Hospital of Tibet Autonomous Region (Tibet, China).

To assess the cell viability, a CCK-8 assay was performed. Briefly, the cells were detached from cell culture dishes, counted and diluted to a concentration of 5×10⁴ cells/ml in a single-cell suspension and then seeded into a 96-well plate at 5.0×10³ cells/well. The cells were cultured and then treated as stated above, dependent on their assigned group. At the end of each experiment, 10 μl CCK-8 reagent (Beyotime Institute of Biotechnology) was added into each well and incubated at 37°C for 1 h, and the optical density was assessed using a microplate reader (Microplate Autoreader EL311; Bio-Tek Instruments, Inc., Winooski, VT, USA) at 450 nm. The experiments were performed in duplicates of five wells for each group and repeated three times.

Nude mice were purchased from the Experimental Animal Center, China Medical University (Shenyang, China). Each group of cells was detached from the cell culture dishes, counted and adjusted to a concentration of 2×10⁷ cells/ml, then 0.2 ml cell suspension was injected into the axilla of each nude mouse. The tumor formation was recorded by measuring the maximum vertical diameters of the length (a) and width (b) every three days. The tumor volume was calculated using the following formula: V (mm³) = 0.5×a×b². The growth curve of the tumor was plotted. After four weeks, the nude mice were sacrificed by intraperitoneal injection of 10% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and the final volume and weight of the tumor xenografts were recorded. One half of each of the xenograft tumors was fixed in 4% paraformaldehyde, embedded in paraffin and sectioned into 5-μm tissue sections for hematoxylin and eosin (H&E) staining (Beijing Solarbio Science & Technology Co., Ltd.). The remaining half of the tumor was immediately frozen in liquid nitrogen and stored at −80°C until further use.

To detect changes in the levels of cell apoptosis in vitro, the Annexin V/propidium iodide (AV/PI) apoptosis detection kit (KeyGen Biotech, Nanjing, China) was used according to the manufacturer's instructions. Briefly, the cells were centrifuged at 1,000 x g for 5 min and the supernatant was discarded. Subsequently, 500 μl binding buffer was added into the tube to re-suspend the cells and 5 μl Annexin V-fluorescein isothiocyanate and propidium iodide were added. The cells were then incubated at room temperature in the dark for 10 min and subjected to flow cytometry (FACSCalibur; BD Biosciences, Mountain View, CA, USA) at a wavelength of 488 nm and an output power of 100 mW. The data were analyzed using the software included in the machine package (Cell Quest; BD Biosciences).

Total cellular RNA was isolated using TRIzol reagent (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions and reverse transcribed into cDNA using a TianScript cDNA first strand cDNA synthesis kit (Tiangen Biotech) according to the manufacturer's instructions. The reaction mix consisted of 1 μg total RNA, 1 μl oligo (dT)₁₅ and random primer, 2 μl deoxyribunucleotide triphosphate and 2 μl double distilled (dd)H₂O with a total volume of 14.5 μl. Following denaturation at 70°C for 5 min, the mixture was placed on ice immediately. Following a short vortex spin, 0.5 μl RNasin (Tiangen Biotech) and 1 μl Moloney murine leukemia virus reverse transcriptase (Bioteke Corporation, Beijing, China) were added into the mixture for incubation at 42°C for 50 min. To terminate the reaction, the reaction mixture was heated at 95°C for 5 min. The primers used for RT-PCR are shown in Table I. The PCR amplification was performed with a 1 μl DNA template, 1 μl of each primer, 10 μl 2X Taq PCR master-mix (Tiangen Biotech) and 8 μl ddH₂O. The PCR cycles were set at an initial 95°C for 5 min and 30 cycles of 95°C for 20 sec, 58°C for 20 sec and 72°C for 30 sec, followed by a final step at 72°C for 5 min. The PCR products were separated by electrophoresis on 1% agarose gels (Biowest, Madrid, Spain), images were captured and levels of β-actin expression were used as the internal control for gray scale analysis (Gel Image System version 4.0; Tanon Science & Technology, Shanghai, China).

Total cellular protein was extracted with a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and the concentration of these protein samples was quantified using the bicinchoninic acid method (Beyotime Institute of Biotechnology). Protein lysates with 40 μg protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). Following blocking with 5% fat-free milk for 1 h, the membranes were incubated with diluted primary antibodies at 4°C overnight. Rabbit polyclonal anti-human β-defensin 2 (hBD-2; cat. no. sc-20798) was used at a dilution of 1:2,000, rabbit polyclonal anti-TLR4 (cat. no. sc-10741) was used at 1:100 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit poly-clonal anti-tumor necrosis factor-α (TNF-α; cat. no. bs-0078R) was used at 1:500, rabbit polyclonal anti-interleukin (IL)-1β (cat. no. bs-0812R) was used at 1:800, rabbit polyclonal anti-IL-6 (cat. no. bs-0379R) was used at 1:800 and rabbit polyclonal IL-12 (cat. no. bs-10641R) was used at 1:500 (Bioss, Beijing, China). Following an overnight incubation, the membranes were washed with Tris-buffered saline-Tween 20 (TBS-T; Beijing Solarbio Science & Technology Co., Ltd.) three times and then further incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. A0208; Beyotime Institute of Biotechnology) at a dilution of 1:1,000 for 1 h. The membranes were then washed again with TBS-T and incubated with enhanced chemiluminescence solution (Pierce Biotechnology, Inc., Rockford, IL, USA) for up to 5 min to develop a color reaction in the dark against an X-ray film (Fujifilm, Shanghai, China). The antibody on the membrane was stripped using stripping buffer (Beyotime Institute of Biotechnology) and the membranes were re-blotted with rabbit polyclonal anti-β-actin antibody (1:1,000 dilution; cat. no. WL0001; Wanleibio, Shenyang, China) as an internal antibody using the same procedure.

Values are presented as the mean ± standard error of the mean and Student's t-test was performed to compare the data between the experimental group and the controls. P<0.05 was considered to indicate a statistically significant difference. SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) was used to analyze the data.

In the present study, the effects of CD14 knockdown were initially assessed with regard to the regulation of gastric cancer cell viability. As compared with the WT group, the cell viability of the LPS cells was significantly increased (P<0.05), whereas viability of the stable CD14-knockdown cells exhibited a slight increase in viability (P<0.05), even following LPS treatment (P>0.05; Fig. 1). The results suggested that CD14 is important in preserving cell viability following treatment with LPS.

The volume of tumors was measured to examine the effect of CD14 knockdown on the growth of tumor xenografts (Fig. 2A). Compared with the WT group, no significant difference was identified in the tumor volume in the sh-CD14 group (P>0.05). By contrast, the tumor volume was markedly increased in the LPS group. However, from day 21, the growth of the tumors gradually slowed down in the sh-CD14 + LPS group, while tumors remained slightly larger than in the WT group, although no statistically significant difference was identified (P>0.05). On day 27 of the experiment, the tumor xenograft tissues were collected and weighed (Fig. 2B). The results revealed that the tumor mass in the WT + LPS group was significantly higher than that in the WT group (P<0.05). However, no statistically significant differences were identified between the WT group and the sh-CD14 and sh-CD14 + LPS groups (P>0.05 for the two groups). H&E staining revealed that the nucleoplasm was larger in the WT + LPS group, exhibiting active cell division and proliferation (Fig. 2C). However, tumor xenograft formation and growth of the sh-CD14 and sh-CD14 + LPS groups were similar, but obviously different from those in the WT group, indicating that LPS may promote the growth of tumor xenografts; however, CD14 had a non-negligible role in mediating LPS signal transduction.

Following Annexin V/propidium iodide staining, the apoptotic rate in each group was determined using flow cytometry (Fig. 3). Compared with the control group (30.13±5.03%), no signifi-cant differences were identified in the apoptotic rate in the sh-CD14 group (30.39±0.82%); however, the rate was markedly reduced in the LPS group (16.88±3.10%). In contrast to that in the WT + LPS group, the apoptotic rate was increased in the sh-CD14 + LPS group (42.47±5.00%), but it remained lower than that in the control group, suggesting that LPS was able to inhibit the apoptosis of gastric cancer cells, and in addition, CD14 was important in the process of apoptosis.

The effects of CD14 knockdown on the regulation of inflammatory gene expression in gastric cancer cell xenografts were assessed. The expression of TNF-α, IL-1β, IL-6, IL-12 and hBD-2 mRNA in each group is shown in Fig. 4. Compared with the WT group, the sh-CD14 group exhibited a decrease in gene expression, but the difference was not statistically significant (P>0.05). The expression of these genes was induced following LPS treatment in the LPS group (P<0.05). Compared with the LPS group, there was a decrease in the expression of these genes in the sh-CD14 + LPS group (P<0.05). The levels of protein expression were in accordance with those of mRNA expression (Fig. 4).

As CD14 binds with TLR4 to form the LPS receptor, the TLR4 expression in the gastric cancer cell xenograft tumors was assessed. Compared with the WT group, the expression of TLR4 mRNA was significantly lower in the sh-CD14 group (P<0.05). Additionally, compared with the LPS group, the expression of TLR4 mRNA was lower in the sh-CD14 + LPS group (P<0.05; Fig. 5A). The expression levels of TLR4 protein were in accordance with those of mRNA expression (Fig. 5B).