Contributed equally
Cluster of differentiation 14 (CD14) protein functions as a co-receptor with either the Toll-like receptor TLR4 or MD-2 in the detection of bacterial lipopolysaccharide (LPS) and plays a role in the innate immune system. Recently, it was shown to have effects on the regulation of epithelial-mesenchymal transition (EMT). Thus, the present study investigated the effects of CD14 knockdown on the regulation of gastric cancer cell EMT and invasive capacity following treatment with or without LPS. Knockdown of CD14 expression using CD14 shRNA in MGC-803 cells significantly enhanced E-cadherin expression, but reduced N-cadherin and vimentin expression in both LPS-treated and untreated cells. Morphologically, the phenotype of LPS-treated CD14-knockdown cells was altered to a sporadic long spindle shape. Moreover, TNF-α-treated cells were further elongated, connections between cells were reduced, the gap between the cells was increased and the cells were transformed into mesenchymal cells. Furthermore, the invasive capacity of CD14-knockdown cells was significantly lower compared to that of the negative control shRNA-transfected MGC-803 cells. LPS-treated CD14-knockdown cells had significantly lower levels of tumor cell invasive ability when compared to the LPS-treated parental MGC-803 cells. However, addition of TNF-α to LPS-treated CD14-knockdown cells significantly increased tumor cell invasion. This study demonstrated that CD14 promoted tumor cell EMT and invasion through TNF-α, whereas knockdown of CD14 expression inhibited gastric cancer cell invasion and EMT.
Gastric cancer is a significant worldwide health issue, accounting for approximately one million new cases and more than 700,000 cancer-related deaths in 2008 (
CD14 also affects the apoptosis of cancer cells and regulates cell cycle distribution through nuclear transcription factors (
Gastric cancer cell lines (SGC-7901, MGC-803, BGC-823 and MKN-28) were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/l streptomycin (Solarbio, Beijing, China) at 37°C in a humidified incubator with 5% CO2 and 95% air. The cells were passaged every 2–3 days with 0.05% trypsin-EDTA.
Total RNA was extracted from cultured cells using TRIzol reagent, and reversely transcribed into cDNA using the cDNA First Strand synthesis kit (both from Beijing Tiangen Company, Beijing, China) according to the manufacturer’s instructions. PCR was performed in a 20 μl volume containing 1 μl of cDNA template, 1 μl of upstream and downstream primers, 10 μl of 2X TaqPCR Master Mix (Beijing Tiangen Company) and 7 μl of water from the Ultrapure Water Polishing System (Heal Force, Hong Kong, China). The primers used in this study are listed in
Total cellular protein was extracted from the cells in RIPA lysis buffer, and the protein concentration was quantified using the BCA method. Protein lysis containing 40 μg protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were then incubated with 5% skim milk solution in phosphate-buffered saline (PBS) for 50 min and then with the primary antibody (i.e., CD14 at a dilution of 1:500; TNF-α at 1:1,000; TGF-β1 at 1:500; E-cadherin at 1:800; N-cadherin at 1:1,000; vimentin at 1:600) at 4°C overnight. Anti-TNF-α antibody was purchased from R&D Systems (Minneapolis, MN, USA), anti-CD14, anti-vimentin and anti-TGF-β1 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and anti-TNF-α, anti-E-cadherin and anti-N-cadherin antibodies were obtained from Abcam (Cambridge, MA, USA). On the following day, the membranes were washed with PBS/Tween-20 and then incubated with a secondary antibody at a dilution of 1:4,000 for 45 min at 37°C. Following washing with PBS-Tween-20 in triplicate, the membranes were incubated with enhanced chemiluminescence (ECL) substrate for visualizing the positive protein bands following expose to X-ray film. The membranes were then stripped and re-blotted with anti-β-actin antibody as the internal control.
In the present study, we designed 4 siRNA primers to knockdown CD14 expression and 1 unrelated interference sequence according to a previously published method (
The stable CD14-knockdown and negative control cells were cultured routinely in RPMI-1640 media containing 10% FBS, 100 U/ml penicillin, 100 mg/l streptomycin and 400 μg/ml G418. Cells at a logarithmic growth phase were then treated with lipopolysaccharides (LPS at 1 μg/ml) for 4 h. The cells were grouped for the following experiments. The treatment groups were as follows: control cells, sh-CD14-alone cells, LPS-alone cells, LPS + sh-CD14 cells, LPS + sh-CD14 + TNF-α cells.
To determine the effects of CD14 knockdown on the invasive ability of the cells with or without LPS treatment, we inoculated cells (2×105/well) into the upper chamber of Transwell plates (Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). In the lower chamber, RPMI-1640 medium containing 10% FBS was added, and the cells were cultured for 48 h. The cells remaining on the surface of the upper chamber were removed using cotton swaps, and the cells that had invaded onto the bottom surface were fixed with 40 ml/l formaldehyde for 15 min and stained with hematoxylin for 2 min. The cells were evaluated and counted under a light microscope. Five fields were chosen for each well, and the numbers of invaded cells were recorded.
Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The experimental data are expressed as means ± SD. The Student’s t-test was used for comparison between 2 groups. P<0.05 was considered to indicate a statistically significant result.
In the present study, we assessed CD14 expression in the different gastric cancer cell lines using semi-quantitative RT-PCR and western blotting. We found that the highest levels of CD14 mRNA and protein were detected in MGC-803 cells, followed by MKN-28 and BGC-823, whereas SGC-7901 had the lowest levels of CD14 mRNA and protein (
To assess the role of CD14 protein in gastric cancer, we used CD14 shRNA to knockdown CD14 expression in the MGC-803 gastric cancer cell line and negative control shRNA was used as a control. Following 48 h of gene transfection, green fluorescent-positive cells were identified as CD14-shRNA. After 4 weeks of G418 selection, 90% of the cells produced green fluorescence, indicating that cells achieved stable transfection of CD14-shRNA. Expression of CD14 at the mRNA and protein levels was then detected in the stable cells, and the data showed that the levels were reduced by 72 and 63%, respectively, when compared to the control cells (
Parental MGC-803 cells exhibited prismatic or polygonal morphology with adherent growth (
We further confirmed the effects of CD14 knockdown on gastric cancer cells. We found that CD14 knockdown increased expression of E-cadherin protein (P<0.05), but reduced expression of N-cadherin and vimentin proteins (P<0.05). Since LPS was able to activate CD14 protein, we treated these cells with LPS for 4 h and found that LPS treatment of parental MGC-803 cells significantly reduced E-cadherin expression (P<0.01), whereas N-cadherin and vimentin expression were significantly increased (P<0.01) compared to the untreated cells, indicating that LPS promotes EMT in gastric cancer cells. However, in the stable CD14-knockdown cells, LPS treatment significantly increased E-cadherin expression (P<0.05), while N-cadherin and vimentin expression was significantly reduced (P<0.05) compared to the cells treated with LPS only, indicating that in the event of lack of CD14 expression, LPS treatment does not induce EMT (
We determined the effects of CD14 knockdown on expression of TNF-α and TGF-β. Compared to LPS-treated parental MGC-803 cells, expression of TNF-α and TGF-β in the LPS-treated CD14-knockdown cells was significantly reduced (P<0.05). Moreover, expression of TNF-α and TGF-β was significantly higher in the LPS and TNF-α-treated CD14-knockdown cells than that of the LPS-treated parental shRNA-transfected cells (
LPS treatment of the CD14 knockdown MGC-803 cells resulted in a higher expression of E-cadherin and lower expression of N-cadherin and vimentin when compared to the LPS-treated negative control shRNA-transfected cells (
Tumor cell EMT usually results in cell migration and invasion. Therefore, we assessed tumor cell invasion capacity following CD14 knockdown and LPS treatment. Indeed, the invasive capacity of CD14-knockdown cells was significantly lower than that of the negative control shRNA-transfected MGC-803 cells (P<0.01;
Cancer invasion and metastases contribute to advanced stage disease and increased cancer-related deaths. Molecularly, they are the consequence of multiple factorial interactions. For cancer metastases, induced tumor cell migration and invasion through cell connection disruption are essential steps. Although EMT is a normal physiological process during embryonic development and organ formation or even during fibrosis, induction of tumor cell EMT can cause tumor migration, invasion and distant metastasis (
LPS is the main component of endotoxin in Gram-negative bacteria, e.g.,
TNF-α, an important inflammatory factor, was reported to induce EMT and promote tumor cell migration and invasion through stabilization of Snail and the NF-κB pathway (
In summary, the present study is a proof-of-principle study to demonstrate the role of CD14 in mediating the EMT and invasion of gastric cancer cells
This study was supported in part by a grant from the National Natural Science Foundation of China (no. 81060165).
Knockdown of CD14 in gastric cancer cell lines. (A) Expression of CD14 in gastric cancer cells analyzed using RT-PCR and western blotting. P<0.05, compared to CD14 expression in the SGC-7901 cells (n=3). (B) Stable knockdown of CD14 expression in gastric cancer MGC-803 cells as detected using RT-PCR and western blotting. P<0.01, compared to the negative control shRNA-transfected cells (n=3). CD14, cluster of differentiation 14.
Effects of CD14 knockdown on gastric cancer cell morphology. (A) Cell morphology. (a) The negative control shRNA-transfected cells; (b) CD14 shRNA-transfected cells; (c) LPS-treated negative control shRNA-transfected cells; (d) LPS-treated CD14 shRNA-transfected cells; (e) LPS and TNF-α-treated CD14 shRNA-transfected cells (x100 magnification). (B) Tumor cell invasion. **P<0.01 compared to the negative control shRNA-transfected cells (group A); #P<0.05 compared to the CD14 shRNA-transfected cells (group B); @@P<0.01 compared to LPS-treated negative control shRNA cells (group C); &&P<0.01 compared to the LPS and TNF-α-treated CD14 shRNA-transfected cells (group E). CD14, cluster of differentiation 14.
Effect of CD14 knockdown on the regulation of gene expression. Expression of (A and B) E-cadherin, (C and D) N-cadherin, (E and F) vimentin, (G and H) TNF-α and (I and J) TGF-β as determined using RT-PCR and Western blot analysis, respectively. The cells with CD14 knockdown were treated with or without LPS (see Materials and method) and were then subjected to RT-PCR and Western blot analysis of gene expression.
Effect of TNF-α treatment on the regulation of E-cadherin, vimentin and N-cadherin expression. Expression of (A and B) E-cadherin (C and D) N-cadherin and (E and F) vimentin as determined using RT-PCR and Western blot analysis, respectively. Cells with CD14 knockdown were treated with or without LPS (see Materials and methods) and were then subjected to RT-PCR and Western blot analysis of gene expression. P-value, compared to the control cells.
PCR primers used in the present study.
Gene | Sequences | Expected PCR product size (bp) |
---|---|---|
CD14 | 5′-TCAGAGGTTCGGAAGACTTATCG-3′ |
239 |
E-cadherin | 5′-ATGCCGCCATCGCTTACAC-3′ |
285 |
N-cadherin | 5′-CAACACACTCGCAGACGCTCA-3′ |
228 |
Vimentin | 5′-CAACACACTCGCAGACGCTCA-3′ |
160 |
TNF-α | 5′-GTCTCCTACCAGACCAAGGTCAAC-3′ |
221 |
TGF-β | 5′-CAACAATTCCTGGCGATACC-3′ |
136 |
CD14, cluster of differentiation 14.