Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion

Human breast cancer cell line MDA-MB-231 was purchased from the cell bank of Academia Sinica (Taipei, Taiwan). MDA-MB-231 was grown without antibiotics in 5% CO₂ at 37°C in RPMI-1640 (Gibco, CA, USA) containing 10% FBS.

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) and the first-strand cDNA was synthesized according to the manufacturer's instructions using 4 μg total RNA with an oligo-dT primer and the myeloblastosis virus (MLV) reverse transcriptase (Promega, WI, USA). The PCR primers for TLRs (from TLR1 to TLR10) and GAPDH were intron-spanning, and are listed in Table 1. PCR products were analyzed on 1-2% (wt/vol) agarose gels containing 0. 5 μg/ml ethidium bromide and were visualized under UV light.

Real-time RT-PCR was performed to detect TLRs gene expression. The 50 μl reaction mixture contained 45 μl DEPC-H₂O, 1.0 μl cDNA (1:100 dilution), 2.0 μl (10 μM) of each primer and freeze-dried powder of the AccuPower Greenstar^® qPCR premix. The thermal cycle profile for PCR was as follows: 94°C for 5 min, 40 cycles of PCR (94°C for 30 sec; 55°C for 30 sec; 72°C for 30 sec). The fluorescence was digitally collected after each cycle of 72°C for 30 sec. After PCR, the samples were subjected to a temperature ramp with continuous fluorescence monitoring for melting curve analysis. BIONEER Exicycler™ analysis software (Bioneer Corp., Daejeon, Korea) was used to obtain the Ct values. 2^{-ΔΔ CT} method [16] was used to analyze the relative expression of each TLR in MDA-MB-231.

To detect the cell protein expression of TLRs, 10⁶ cultured MDA-MB-231 were prefixed and permeabilized. Then, the cells were stained with 3 μl purified anti-human TLR4 antibody (Santa Cruz Biotechnology, CA, USA) at 4°C for 30 min away from light. After washing twice with 1×PBS, the cells were incubated with 2 μl PE-conjugated goat anti-rabbit IgG mAb (Santa Cruz Biotechnology) at 4°C for 30 min away from light, followed by an additional two washes with 1×PBS. Finally, the stained cells in 500 μl 1×PBS were analyzed by using a flow cytometer (FACScalibur; Becton Dickinson (BD), NJ, USA), and the data were processed with BD CellQuest software. The negative control was performed by omitting the anti TLR4 antibody.

Cells cultured overnight were fixed with alcohol for 30 min and blocked in 1×PBS (pH 7.4) solution with 3% BSA overnight at 4°C in a hydrated box. Anti-TLR4 antibody was added at a 1:100 dilution (Santa Cruz Biotechnology) and allowed to incubate overnight at 4°C in a hydrated box. After washing three times, fluorescent secondary antibody (Santa Cruz Biotechnology) was added at a 1:100 dilution. The cells were again washed three times with 1×PBS, and counter-stained with DAPI. Fluorescence was analyzed by fluorescence microscope (DMI4000B; Leica, IL, USA). Adobe Photoshop 9.0 software (CA, USA) was used for subsequent image processing.

Cells were transiently transfected with a GFP expressing plasmid pGsil-1 (Genesil, Wuhan, China) containing silencing RNA (siRNA) directed against TLR4. The three pieces of small interfering oligonucleotide specific for human TLR4 have been listed in Table 2 . Briefly, 2×10⁵ cells were seeded in 6-well dishes and cultured overnight until 60% to 70% confluency was reached. Transfections were performed using Lipofectamine™ 2000 reagent (Invitrogen) per the manufacturer's instructions. Cells were transfected with 4 μg plasmid DNA (TLR4AsiRNA, TLR4BsiRNA, TLR4CsiRNA, vector pGenesil-1 and ScrambledsiRNA) using 8 μl transfection reagent. After 48 h of transfection, fluorescence of cells was observed by a fluorescence microscope. Then, cells were seeded for FCM and immunofluorescence assay. Supernatant was collected to test the inflammatory cytokines secreted by the cells.

Cells were seeded into 96-well culture plates (6×10³/well, 5 wells repeated), allowed to adhere overnight, and then transfections were performed according to the manufacturer's instructions. After 48 h, the transfected cells were collected (0 h) or allowed to continue in culture for 24 h, 48 h, or 72 h. At the end of each treatment, cells were incubated with 5 mg/mL MTT (Sigma Chemical Co., MO, USA) for 4 h and then mixed with dimethyl sulfoxide after the supernatant was removed. The dye absorption (A) was quantitated using an automatic microplate spectrophotometer (340 st; Anthos Zenyth, Salzburg, Austria) at 490 nm.

IL-6 and IL-8 presence in the supernatant of transfected cells were detected according to the instruction of human inflammatory cytokine kit (BD™ Cytometric Bead Array (CBA)). FACScan flow cytometer (BD) was used to analyze samples.

GraphPad Prism software (CA, USA) was used to perform statistical comparisons between different values. Data were expressed as the means ± standard deviation (SD) with n = 3. Statistical significances were determined by Student's t- test and ANOVA, differences were considered significant at a P value of less than 0.05.

As TLRs have been identified in some tumor cells, we sought to detect if they were expressed in the human breast cancer cell line MDA-MB-231. Qualitative RT-PCR analysis revealed that MDA-MB-231 expressed mRNA of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 (Figure 1A). Real-time PCR analysis revealed the relative expressions of each TLR examined. The expression of TLR3 was normalized to 1.0, as it was expressed the most weakly. TLR4 was 5-fold higher than TLR3, while other TLRs were expressed between 1- and 4-fold higher than TLR3 (Figure 1B). By FCM detection, we were able to examine the different protein expression levels of the TLRs, TLR4, TLR6, TLR7 and TLR5 were expressed moderately; the other TLRs were expressed weakly or unexpressed. Again, TLR4 protein level was the highest out of TLR1-TLR10 (Figure 1C). Collectively, these results demonstrated that MDA-MB-231 expressed all the TLRs examined (TLR1-TLR10) and TLR4 was expressed highest. TLR4 was strategically selected to investigate its function on the growth and progression of MDA-MB-231 in subsequent studies.

To study the biological role of TLR4 in the progression of human breast cancer cell line MDA-MB-231, we constructed pGenesil-1 plasmid vectors expressing three different siRNAs directed against TLR4 [GenBank: NM_138554.3] to selectively reduce TLR4 gene expression in MDA-MB-231. The regions have no significant homology to other coactivators or sequences in the human genome database. The vector, TLR4AsiRNA, TLR4BsiRNA, TLR4CsiRNA and ScrambledsiRNA were transfected into MDA-MB-231. After 48 h, the transfected cells appeared to fluoresce green under the fluorescence microscope. Transfection efficiency reached about 70%. From RT-PCR we could see that there were different reductions in TLR4AsiRNA, TLR4BsiRNA, TLR4CsiRNA transfected cells (Figure 2A). Figure 2B showed us that the decreased expression of TLR4 at mRNA levels for TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA was 74.8 ± 9.2%, 55.2 ± 6.7% and 63.0 ± 8.3% as compared to vector control (P < 0.05). However, no significant difference was observed in siRNA control (P > 0.05). As shown in Figure 2C, analysis of the transfected cells for TLR4 expression via FCM demonstrated that specific reductions at protein level for TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA was 53.0% ± 2.9%, 37.9% ± 3.7% and 46.7% ± 4.6% as compared to vector control (P < 0.05). No obvious difference was seen in siRNA control (P > 0.05). Human beast cancer cell line MDA-MB-231 showed that siRNA-directed knockdown of the TLR4 gene was specific. TLR4AsiRNA was the most efficient recombinant plasmid in silencing TLR4 and it was chosen for use in subsequent functional assay.

Real-time PCR had demonstrated a specific reduction at mRNA level for TLR4AsiRNA. We further analyzed the effects of TLR4AsiRNA on TLR4 protein expression in MDA-MB-231 using immunostaining with anti-TLR4 antibody. Red fluorescence of TLR4 staining under the fluorescence microscope was drastically reduced by TLR4AsiRNA in comparison to vector control. No obvious difference was seen in siRNA control (Figure 3A). To access the potential effects of TLR4AsiRNA-mediated TLR4 silencing on cell proliferation and survival, MTT analysis was performed on the cells cultured 0 h, 24 h, 48 h, and 72 h following 48 h of transfection. Targeting of TLR4AsiRNA against TLR4 effected the proliferative ability of MDA-MB-231 (Figure 3B). The proliferative rate was significantly decreased according to the time of culture after transfection with TLR4AsiRNA compared with vector control; no significant difference was observed in siRNA control (P > 0.05). The biological consequences caused by TLR4 silencing may be a result of changes in TLR4-mediated signaling and subsequent downstream functions. Because increased TLR4 activates TLR4/MyD88 signaling and subsequent downstream functions [17], we decided to examine the status of the TLR4-related inflammatory cytokines in MDA-MB-231 with TLR4 gene knockdown. Analysis of FCM revealed that IL-6 and IL-8 were markedly depressed in the supernatant of silenced cells. The inhibition ration of cytokine IL-6 and IL-8 was 47.8 ± 3.9% and 48.3 ± 4.1% respectively when compared with vector control (P < 0.05), no significant difference was seen in siRNA control (Figure 3C and Figure 3D). These results suggested that decreased TLR4 levels in tumor cells might endow cells with attenuated growth and survival capacity.