Open Access

Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells

  • Authors:
    • Yating Liu
    • Tao Li
    • Yuanhong Xu
    • Enjun Xu
    • Min Zhou
    • Baolong Wang
    • Jilong Shen
  • View Affiliations

  • Published online on: March 16, 2016     https://doi.org/10.3892/ol.2016.4338
  • Pages: 3054-3060
  • Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Toll-like receptors (TLRs) are key factors in the innate immune system and initiate an inflammatory response to foreign pathogens, such as bacteria, fungi and viruses. TLR4-mediated signaling has been implicated in tumor cell proliferation and apoptosis in numerous cancers. The present study aimed to investigate the biological effect of TLR4 on the proliferation and apoptosis of human liver cancer cells and the mechanisms responsible for the regulation of cellular responses following TLR4 gene knockdown. Three TLR4 small interfering (si)RNA constructs, consisting of TLR4‑siRNA‑1, TLR4‑siRNA‑2 and TLR4‑siRNA‑3, were transiently transfected into HepG2 cells using Lipofectamine 2000. TLR4 knockdown was confirmed using reverse transcription‑polymerase chain reaction and western blotting. The effect of the TLR4 siRNA on tumor cell proliferation was monitored by methyl thiazolyl tetrazolium assay and cell apoptosis was observed by flow cytometry. The expression of TLR4‑associated proteins, consisting of myeloid differentiation primary response 88 (MyD88), Toll‑interleukin‑1R‑domain‑containing adapter‑inducing interferon‑β (TRIF), interferon regulatory factor‑3 (IRF3), nuclear factor (NF)‑κB, NF‑κB inhibitor α (IκBα), phosphorylated IκBα (p‑IκBα), extracellular signal‑regulated kinase (ERK) and c‑Jun N‑terminal kinase (JNK), was detected by western blot analysis. TLR4‑siRNA‑1 had the strongest knockdown effect and inhibited TLR4 messenger RNA and protein expression. TLR4 knockdown with TLR4‑siRNA‑1 reduced cell proliferation and promoted cell apoptosis. MyD88, TRIF, IRF3, IκBα, JNK and ERK were markedly suppressed in the cells transfected with TLR4 siRNA. However, nuclear expression of NF‑κB and p‑IκBα increased in HepG2 cells with TLR4 gene knockdown. The present study revealed that TLR4‑mediated signaling plays a key role in the proliferation and apoptosis of cultured hepatocarcinoma cells. Therefore, RNA interference-directed targeting of TLR4 may raise the potential of the application of TLR4 knockdown for liver cancer therapy.

Introduction

Hepatocellular carcinoma (HCC) is considered as a malignant cancer originating from liver cells; HCC is currently the fifth most common solid tumor worldwide, and the incidence of HCC increases yearly (1). The incidence and mortality rates of liver cancer are high in urban and rural areas of China (2). Currently, patients with HCC have a poor long-term prognosis, and surgery offers little hope for successful treatment. Consequently, various forms of combination therapy are encouraged in the clinic (3). The lack of an effective treatment has led to a search for a novel treatment strategy, such as gene therapy. Previously, it was suggested that Toll-like receptors (TLRs) are expressed in numerous human tumor cells (4).

TLRs are a family of pattern recognition receptors that recognize pathogen-associated molecular patterns and damage-associated molecular patterns (5). TLRs contain an intracellular domain, which is homologous to the intracellular domain of the mammalian interleukin (IL)-1 receptor, termed the Toll-IL-1R (TIR) function domain. At present, 13 mammalian TLRs have been identified and characterized, termed TLR1 to TLR13, respectively, but 10 human TLRs have been identified and characterized, termed TLR1 to TLR10, respectively (6).

TLR4 plays different roles in different tumor cell types. TLR4 signaling has been found to promote tumor growth (7). TLR4 signaling induced by lipopolysacharide (LPS) or paclitaxel was also found to regulate tumor survival in ovarian cancer (8). LPS stimulated colorectal cancer cell adhesion and invasion through TLR4 and nuclear factor (NF)-κB-dependent activation of the urokinase plasminogen activator system (9). Small interfering (si)RNA-directed targeting of TLR4 inhibited the invasion, survival and tumorigenicity of human prostate cancer cell (10). Triggering the expression of TLR4 on human head and neck squamous cell carcinoma promoted tumor development, promoted the secretion of IL-6, and protected the tumor from immune attack (11).

TLR4 ligation activates at least two signaling pathways, consisting of the myeloid differentiation primary response 88 (MyD88)-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways (12). The former pathway recruits TIR domain containing adaptor protein and MyD88 for NF-κB activation, leading to the upregulation in anti-apoptotic signaling. The MyD88-independent pathway involves TRIF-related adaptor molecule and, instead of MyD88, recruits TRIF and tumor necrosis factor receptor-associated factor 3, leading to the induction of interferon regulatory factor-3 (IRF3) and late NF-κB activation (13). Despite considerable insights into the TLR4 signaling pathway, biological effects of knockdown of TLR4 in HCC remain controversial.

The present study investigated the biological effect of TLR4 on the proliferation and apoptosis of the human liver cancer HepG2 cell line, and investigated the mechanisms responsible for the regulation of cellular responses following TLR4 gene knockdown, to assess its potential in the field of cancer therapy.

Materials and methods

Cell line

The human hepatocellular carcinoma HepG2 cell line was obtained from the cell bank of Anhui Provincial Laboratory of Pathogen and Biology Zoonoses, Anhui Medical University (Hefei, China). HepG2 cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and streptomycin (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C with 5% CO2.

RNA interference assay

The cells were transfected with various siRNA directed against TLR4 (catalog nos.: siRNA1, TLR4-homo-1546; siRNA2, TLR4-homo-1325; siRNA3, TLR4-homo-595), and negative siRNA with a random sequence was used as a control (Shanghai GenePharma Co., Ltd., Shanghai, China). The siRNA sequences are shown in Table I. The cells were seeded at a density of 5×105 cells/well into 6-well dishes and cultured overnight at 37°C with 5% CO2 until the cells reached 70% confluency. The transfections were performed using Invitrogen Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturers protocol.

Table I.

Sequences of TLR4-siRNA and negative siRNA.

Table I.

Sequences of TLR4-siRNA and negative siRNA.

siRNASequence, 5′-3′
TLR4-siRNA-1
  Sense GGGCUUAGAACAACUAGAATT
  Antisense UUCUAGUUGUUCUAAGCCCTT
TLR4-siRNA-2
  Sense CCCACAUUGAAACUCAAAUTT
  Antisense AUUUGAGUUUCAAUGUGGGTT
TLR4-siRNA-3
  Sense CCACCUCUCUACCUUAAUATT
  Antisense UAUUAAGGUAGAGAGGUGGTT
Negative-siRNA
  Sense UUCUCCGAACGUGUCACGUTT
  Antisense ACGUGACACGUUCGGAGAATT

[i] TLR, Toll-like receptor; siRNA, small interfering RNA.

Optimization of transfection conditions

The cells were seeded at a density of 1×105 cells/well into 24-well dishes and cultured overnight at 37°C with 5% CO2 until the cells reached 70% confluency. Then, fluorescein amidite (FAM)-labeled negative control siRNA was transfected into cells using Lipofectamine 2000. The ratios of siRNA to Lipofectamine 2000 used were 0.5:1, 1:1, 1.5:1, 2:1 and 2.5:1 (µl/µl). Following 24 h of transfection, the transfection efficiency was observed using fluorescence microscopy (model no. IX51; Olympus, Melville, NY, USA), and the ratio of the number of fluorescent cells to non-fluorescent cells within 100 cells observed at high magnification was determined to be the transfection efficiency.

Reverse transcription-polymerase chain reaction (RT-PCR)

After 24 h of transfection, Invitrogen TRIzol reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA, according to the manufacturers protocol. Subsequently, 2 µg of total RNA was used as the template for single-strand complementary (c)DNA synthesis, which was performed using a RevertAid First Strand cDNA Synthesis kit (cat no. K1622; Thermo Fisher Scientific, Inc.), according to the manufacturers protocol. The TLR4 primers were as follows: Forward, 5-TGAGCAGTCGTGCTGGTATC-3; and reverse, 5-CAGGGCTTTTCTGAGTCGTC-3. β-actin was used as an internal control and was also amplified, using the following primers: Forward 5-TCCTGTGGCATCCACGAAACT-3′; and reverse, 5-GAAGCATTTGCGGTGGACGAT-3. The PCR conditions were as follows: 95°C for 3 min; 30 cycles at 95°C for 30 sec; 52.9°C for 30 sec; and 72°C for 30 sec. The final extension was performed at 72°C for 10 min. The PCR products were analyzed on 2% (wt/vol) agarose gels containing 0.5 µg/ml ethidium bromide and were visualized under ultraviolet light. Brand density was analyzed and quantified using Molecular Dynamics Image Quant software (GE Healthcare Life Sciences, Chalfont, UK).

Western blot

Subsequent to 48 h of transfection, the cells were washed and lysed in radioimmunoprecipitation assay (RIPA; Beyotime Institute of Biotechnology, Haimen, China) buffer. Phenylmethanesulfonyl fluoride (Thermo Fisher Scientific, Inc.) was added to the RIPA buffer, according to the manufacturers protocol. Protein concentrations were determined on diluted samples using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Equal amounts of protein were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membranes (Beyotime Institute of Biotechnology). The membranes were blocked in 1X Tris-buffered saline (TBS; ZSGB-BIO, Beijing, China) with Tween-20 (TBST; Beyotime Institute of Biotechnology), consisting of 0.05% Tween-20 and 1X TBS (pH 7.4), with 5% nonfat dry milk and incubated with mouse monoclonal anti-TLR4 (dilution, 1:800; cat no.,ab22048; Abcam), mouse monoclonal NF-κB inhibitor α (IκBα; dilution, 1:1,000; cat no. 4814; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse monoclonal phosphorylated-IκBα (p-IκBα; dilution, 1:1,000; cat no. 9246; Cell Signaling Technology, Inc.), rabbit polyclonal MyD88 (dilution, 1:1,000; cat no. BS3521; Bioworld Technology, Inc., St. Louis Park, MN, USA), rabbit polyclonal TRIF (dilution, 1:1,000; cat no. ab13810; Abcam), rabbit polyclonal extracellular signal-regulated kinase (ERK; dilution, 1;1,000; cat no. 9102; Cell Signaling Technology, Inc.), rabbit polyclonal c-Jun N-terminal kinase (JNK; dilution, 1:1,000; cat no. 9252; Cell Signaling Technology, Inc.), rabbit monclonal IRF3 (dilution, 1:1,000; cat no. 4302; Cell Signaling Technology, Inc.), rabbit polyclonal NF-κB (dilution, 1:1,000; cat no. BS3602; Bioworld Technology, Inc.), mouse monoclonal lamin A/C (dilution, 1:2,000; cat no. 4777; Cell Signaling Technology, Inc.) and mouse monoclonal anti-β-actin (dilution, 1:1,000; cat no. TA-09; ZSGB-BIO) antibodies in blocking solution overnight at 4°C. They were later washed three times with TBST at 10 min intervals, incubated with horseradish peroxidase-conjugated anti-mouse goat polyclonal (cat no. ZB-2305) or anti-rabbit goat polyclonal (cat no. ZDR-5306) secondary antibodies (dilution, 1:5,000; ZSGB-BIO) in blocking solution 1 h at room temperature, and washed three times with TBST at 10-min intervals. Signals were visualized by chemiluminescence (ChemiQ3650; Bioshine, Shanghai, China) and quantitated using ImageQuant software.

Cell proliferation assay

The methyl thiazolyl tetrazolium (MTT) assay was used to evaluate cell proliferation subsequent to transfection. The cells were cultured overnight at 37°C with 5% CO2 in 96-well cell plates at a density of 5×103 cells/well, with 5 wells per condition, and cell transfections were performed according to the manufacturer's protocol. Subsequent to 24 h, a sample of the transfected cells was collected as the 0 h sample, while the other cells continued in culture for 24, 48, 72 and 96 h. At the end of each treatment period, 5 mg/ml MTT was added to the culture medium in each well (Sigma-Aldrich, St. Louis, MO, USA) and then incubated for 4 h at 37°C. The supernatant was then removed and cells were mixed with 150 µl/well dimethyl sulfoxide. The absorption was measured using an automatic microplate reader (ELx808; Dynex Technologies, Inc., Chantilly, VA, USA) at 490 nm.

Flow cytometry

A flow-based Annexin V assay was used to measure cell apoptosis after transfection. Briefly, the cells were treated with medium alone or in the presence of siRNA specific for TLR4 or with negative control siRNA or with Lipofectamine 2000 only for 48 h. The cells were washed in PBS, resuspended in 400 µl of ANX-V binding buffer and then stained with 5 µl of Annexin-V-fluorescein isothiocyanate (FITC) for 15 min on ice in the dark, according to the manufacturers instructions. Subsequent to staining, the cells were incubated with 10 µl of propidium iodide (PI) for 5 min on ice in the dark. Analyses were performed with flow cytometry (Becton Dickinson, Bedford, MA, USA). The cells in the FITC-positive fraction were regarded as apoptotic.

Statistical analysis

Data shown are from at least three separate experiments and are expressed as the mean ± standard deviation (SD). Student's t-test and analysis of variance were used to determine the significance of differences between groups. Student-Newman-Keuls method was used as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Transfection condition

FAM-labeled negative control siRNA was transfected into cells with Lipofectamine 2000. Subsequent to 24 h of transfection, the transfection efficiency was observed using fluorescence microscopy. The ratio of siRNA to Lipofectamine 2000 of 1:1 resulted in the highest transfection efficiency of 90.48±1.27% (Fig. 1). Fluorescent particles within the cells indicated that siRNA was transfected successfully into HepG2 cells (Fig. 1C).

Knockdown of the TLR4 gene using siRNA in the HepG2 cell line

To investigate the potential function of TLR4 in hepatocellular carcinoma, siRNAs were used to knockdown endogenous TLR4 gene expression (gene ID, 7099). In total, 3 siRNAs were transfected into HepG2 cells, with medium alone, negative siRNA or Lipofectamine 2000 alone as controls. Subsequent to 24 h, TLR4-siRNA-1 and TLR4-siRNA-3 effectively reduced TLR4 messenger (m)RNA expression in transfected cells (Fig. 2A). The TLR4 mRNA levels were 14.48±2.06, 50.73±7.09, 37.78±3.9, 54.78±7.75, 53.7±6.58 and 51.4±4.89% in cells treated with TLR4-siRNA-1, TLR4-siRNA-2, TLR4-siRNA-3, medium alone, negative control siRNA and Lipofectamine 2000 alone, respectively (Fig. 2B). There was a significant difference between the expression of TLR4 in the TLR4-siRNA-1 group and blank control group (t=8.699; P=0.001) and TLR4-siRNA-3 group and blank control group (t=3.39; P=0.028). The expression in the TLR4-siRNA-2, negative sequence transfection and Lipofectamine 2000 alone groups was not significantly different from the expression in the blank control group (P>0.05).

To study TLR4 protein expression using TLR4-siRNA-1, TLR4-siRNA-1 was transfected into HepG2 cells, with medium, negative siRNA or Lipofectamine 2000 alone acting as controls. Following 48 h, TLR4-siRNA-1 effectively reduced TLR4 protein expression in transfected cells (Fig. 2C). TLR4 protein levels were 5.92±5.82, 25.13±3.04, 20.18±2.93 and 21.43±1.57% in the cells treated with TLR4-siRNA-1, medium alone, negative control siRNA and Lipofectamine 2000 alone, respectively (Fig. 2D). A significant decrease in protein expression was observed in the TLR4-siRNA-1 group compared with the blank control group. The negative sequence transfection and Lipofectamine 2000 alone groups were not significantly different from the blank control group (P>0.05).

Therefore, the present study demonstrated specific siRNA-directed knockdown of the TLR4 gene in the human hepatocellular carcinoma HepG2 cell line, and these data indicate that TLR4-siRNA-1 demonstrated the most efficient silencing of TLR4. Only TLR4-siRNA-1 was selected for use in the subsequent experiments.

Proliferation of the transfected HepG2 cell line following TLR4 gene knockdown

The MTT assay was used to determine the effects of TLR4-siRNA-1-mediated TLR4 silencing on cell proliferation. The cells were cultured for 0, 24, 48, 72 and 96 h subsequent to 24 h of transfection. The proliferative ability of HepG2 cells was reduced by TLR4-siRNA-1 transfection subsequent to culturing for 48 h (Fig. 3). No significant difference was observed among the blank control, negative sequence transfection and Lipofectamine 2000 alone groups (P>0.05).

Apoptosis of the transfected HepG2 cell line following TLR4 gene knockdown

A flow-based Annexin V assay was used to determine the effects of TLR4-siRNA-1-mediated TLR4 silencing on cell apoptosis. TLR4-siRNA-1 promoted apoptosis levels in HepG2 cells subsequent to 48 h transfection (Fig. 4A). The apoptosis rates of the TLR4-siRNA-1, blank control, the negative sequence transfection and Lipofectamine 2000 alone groups were 8.91±1.00, 2.48±0.29, 2.71±0.17 and 2.59±0.29%, respectively (Fig. 4B). The apoptosis rate of TLR4-siRNA-1 group compared with three control groups showed statistical significance (P<0.05). No significant difference was observed among the 3 control groups (P>0.05).

Knockdown of the TLR4 gene by siRNA blocked downstream signaling in the HepG2 cell line

The biological consequences of TLR4 silencing may be a result of changes in TLR4-mediated signaling and subsequent downstream functions. The present study examined the status of the TLR4-associated proteins, including MyD88, TRIF, IRF3, IκBα, p-IκBα, NF-κB, ERK, and JNK in HepG2 cells with TLR4 gene knockdown. MyD88, TRIF and IRF3 were markedly suppressed in the cells transfected with TLR4 siRNA when compared with control cells (Fig. 5). Furthermore, decreased activity of the IκBα, JNK and ERK signaling pathways was observed in HepG2 cells following TLR4 gene knockdown. Nuclear expression of NF-κB and p-IκBα increased in HepG2 cells with TLR4 gene knockdown.

Discussion

TLR mediates the inherent immune inflammatory response, although more studies consider the association between TLR activation, uncontrolled inflammation and tumor development (1417). TLR4 and other TLRs have been detected in throat, breast, colorectal, gastric, prostate and lung cancer cell lines (15). The silencing of TLR4 signaling pathways in cancer cells may reduce the risk of tumor formation (16). LPS-induced TLR4 signaling in cancer cells promoted cell survival and proliferation in HCC (17). Although TLR4 was hypothesized to play an important role in the initiation and progression of HCC, little is known about the interaction between TLR4 and disease progression. In the present study, the biological effect of TLR4 on cell growth and survival was investigated.

The best transfection condition was tested to ensure that siRNA was transfected into HepG2 cells successfully using Lipofectamine 2000. TLR4 mRNA and protein were respectively detected by RT-PCR and western blot analysis subsequent to transfection of the HepG2 cells with siRNA. The present study confirmed the TLR4-siRNA-1 was the most efficient siRNA for silencing TLR4, suggesting that HepG2 cells transfected with TLR4-siRNA-1 using Lipofectamine 2000 was feasible and effective. This provides a reliable tool to study the biological effect of TLR4 on liver cancer cells.

The results showed that TLR4 knockdown inhibits human liver cancer cell proliferation and promotes cell apoptosis in vitro. TLR4 may be associated with the biological behavior of HCC, such as proliferation and apoptosis; TLR4-mediated cancer growth appears to be an important factor in tumor progression.

Suppression of the TLR4 downstream signaling molecules MyD88, TRIF and IRF3 was observed in TLR4 siRNA-transfected HepG2 cells, but not in negative siRNA/Lipofectamine alone-transfected cells. This indicated that all the observed phenotypic changes in these cells were mediated by suppressing the TLR4 action and its downstream molecules. Therefore, it has been demonstrated that TLR4-mediated signaling stimulated MyD88/TRIF/IRF pathways and protected cancer cells from death.

The ERK signaling pathway is involved in the regulation of cell survival and cell death (18). Increased ERK activity confers an aggressive phenotype in cancer cells and is correlated with decreased patient survival rates, whereas blockage of ERK activation may inhibit cancer growth in human HCC (19,20). The role of JNK in liver disease remains unclear (21). JNK is rapidly activated to participate in liver regeneration subsequent to partial hepatectomy (22). The present results indicated that ERK and JNK activity was decreased by suppression of TLR4 activity, which may exert a pro-apoptotic effect on HepG2 cells.

The important role of NF-κB in cancer development came through its abnormal activation by numerous oncogenes. By inhibiting mRNA transcription of caspase 3, caspase 7, procaspase 6 and procaspase 9, NF-κB activation inhibits the death receptor and mitochondrial pathways of apoptosis (23). NF-κB inactivation increased the expression of IL-6 and the incidence of HCC (24). NF-κB inactivation in keratinocytes enhanced the development of squamous cell carcinoma (25). A previous study has found that the inhibition of NF-κB activation promoted cancer cell proliferation in HCC cells (17). The present study found that the activity of NF-κB and p-IκBα increased by suppressing the TLR4 action. These results suggest that NF-κB may play a conflicting role in hepatocellular carcinoma.

The present results provide evidence that TLR4 knockdown inhibits the proliferation of human liver cancer cells and promotes cell apoptosis in vitro through modulation on its downstream signaling pathways in HepG2. The use of systemically delivered TLR4 siRNA may provide a novel treatment for the prevention of cancer progression leading to improved prospects of survival.

The results presented in the present study provide evidence that TLR4 knockdown inhibits human liver cancer cell proliferation and promotes cell apoptosis in vitro through the modulation on the downstream signaling pathways in HepG2. The use of systemically delivered TLR4 siRNA may provide a novel treatment for the prevention of cancer progression, leading to improved survival rate.

Acknowledgements

The authors would like to thank Mr Qingli Luo and Mr He Chen (Anhui Provincial Laboratory of Pathogen and Biology Zoonoses, Anhui Medical University, Hefei, Anhui, China) for providing technical support. The current study was supported by grants from the National Natural Science Foundation of China (grant nos. 30801088 and 81201488) and the Application Research Project of the Ministry of Health (grant no. 28-1-50).

References

1 

Baffy G, Brunt EM and Caldwell SH: Hepatocellular carcinoma in non-alcoholic fatty liver disease: An emerging menace. J Hepatol. 56:1384–1391. 2012. View Article : Google Scholar : PubMed/NCBI

2 

Chen W, Zheng R, Zhang S, Zhao P, Li G, Wu L and He J: The incidences and mortalities of major cancers in China, 2009. Chin J Cancer. 32:106–112. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Yang J, Yan L and Wang W: Current status of multimodal & combination therapy for hepatocellular carcinoma. Indian J Med Res. 136:391–403. 2012.PubMed/NCBI

4 

Muccioli M, Sprague L, Nandigam H, Pate M and Benencia F: Toll-like receptors as novel therapeutic targets for ovarian cancer. ISRN Oncol. 2012:6421412012.PubMed/NCBI

5 

Tang D, Kang R, Carolyn CB, Zeh HJ and Lotze MT: PAMPs and DAMPs: Signal 0s that spur autophagy and immunity. Immunol Rev. 249:158–175. 2012. View Article : Google Scholar : PubMed/NCBI

6 

Kawai T and Akira S: The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nat Immunol. 11:373–384. 2010. View Article : Google Scholar : PubMed/NCBI

7 

Lee CH, Wu CL and Shiau AL: Toll-like receptor 4 signaling promotes tumor growth. J Immunother. 33:73–82. 2010. View Article : Google Scholar : PubMed/NCBI

8 

Szajnik M, Szczepanski MJ, Czystowska M, Elishaev E, Mandapathil M, Nowak-Markwitz E, Spaczynski M and Whiteside TL: TLR4 signaling induced by lipopolysacharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer. Oncogene. 28:4353–4363. 2009. View Article : Google Scholar : PubMed/NCBI

9 

Killeen SD, Wang JH, Andrews EJ and Redmond HP: Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system. Br J Cancer. 100:1589–1602. 2009. View Article : Google Scholar : PubMed/NCBI

10 

Hua D, Liu MY, Cheng ZD, Qin XJ, Zhang HM, Chen Y, Qin GJ, Liang G, Li JN, Han XF and Liu DX: Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion, survival and tumorigenicity. Mol Immunol. 46:2876–2884. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Szczepanski MJ, Czystowska M, Szajnik M, Harasymczuk M, Boyiadzis M, Kruk-Zagajewska A, Szyfter W, Zeromski J and Whiteside TL: Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res. 69:3105–3113. 2009. View Article : Google Scholar : PubMed/NCBI

12 

Tanimura N, Saitoh S, Matsumoto F, Akashi-Takamura S and Miyake K: Roles for LPS-dependent interaction and relocation of TLR4 and TRAM in TRIF-signaling. Biochem Biophys Res Commun. 368:94–99. 2008. View Article : Google Scholar : PubMed/NCBI

13 

Kreuz S, Siegmund D, Rumpf JJ, Samel D, Leverkus M, Janssen O, Häcker G, Dittrich-Breiholz O, Kracht M, Scheurich P and Wajant H: NF-kappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP. J Cell Biol. 166:369–380. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Sipos F, Fűri I, Constantinovits M, Tulassay Z and Műzes G: Contribution of TLR signaling to the pathogenesis of colitis-associated cancer in inflammatory bowel disease. World J Gastroenterol. 20:12713–12721. 2014. View Article : Google Scholar : PubMed/NCBI

15 

Huang B, Zhao J, Unkeless JC, Feng ZH and Xiong H: TLR signaling by tumor and immune cells: A double-edged sword. Oncogene. 27:218–224. 2008. View Article : Google Scholar : PubMed/NCBI

16 

Huang B, Zhao J, Li H, He KL, Chen Y, Chen SH, Mayer L, Unkeless JC and Xiong H: Toll-like receptors on tumor cells facilitate evasion of immune surveillance. Cancer Res. 65:5009–5014. 2005. View Article : Google Scholar : PubMed/NCBI

17 

Wang L, Zhu R, Huang Z, Li H and Zhu H: Lipopolysaccharide-induced toll-like receptor 4 signaling in cancer cells promotes cell survival and proliferation in hepatocellular carcinoma. Dig Dis Sci. 58:2223–2236. 2013. View Article : Google Scholar : PubMed/NCBI

18 

Balmanno K and Cook SJ: Tumor cell survival signalling by the ERK1/2 pathway. Cell Death Differ. 16:368–377. 2009. View Article : Google Scholar : PubMed/NCBI

19 

Schmitz KJ, Wohlschlaeger J, Lang H, Sotiropoulos GC, Malago M, Steveling K, Reis H, Cicinnati VR, Schmid KW and Baba HA: Activation of the ERK and AKT signalling pathway predicts poor prognosis in hepatocellular carcinoma and ERK activation in cancer tissue is associated with hepatitis C virus infection. J Hepatol. 48:83–90. 2008. View Article : Google Scholar : PubMed/NCBI

20 

Gailhouste L, Ezan F, Bessard A, Frémin C, Rageul J, Langouët S and Baffet G: RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo. Int J Cancer. 126:1367–1377. 2010.PubMed/NCBI

21 

Seki E, Brenner DA and Karin M: A liver full of JNK: Signaling in regulation of cell function and disease pathogenesis and clinical approaches. Gastroenterology. 143:307–320. 2012. View Article : Google Scholar : PubMed/NCBI

22 

Schwabe RF, Bradham CA, Uehara T, Hatano E, Bennett BL, Schoonhoven R and Brenner DA: c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration. Hepatology. 37:824–832. 2003. View Article : Google Scholar : PubMed/NCBI

23 

Karin M and Lin A: NF-kappaB at the crossroads of life and death. Nat Immunol. 3:221–227. 2002. View Article : Google Scholar : PubMed/NCBI

24 

Maeda S, Kamata H, Luo JL, Leffert H and Karin M: IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell. 121:977–990. 2005. View Article : Google Scholar : PubMed/NCBI

25 

van Hogerlinden M, Auer G and Toftgård R: Inhibition of Rel/Nuclear Factor-kappaB signaling in skin results in defective DNA damage-induced cell cycle arrest and Ha-ras-and p53-independent tumor development. Oncogene. 21:4969–4977. 2002. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

May-2016
Volume 11 Issue 5

Print ISSN: 1792-1074
Online ISSN:1792-1082

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Liu Y, Li T, Xu Y, Xu E, Zhou M, Wang B and Shen J: Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells. Oncol Lett 11: 3054-3060, 2016.
APA
Liu, Y., Li, T., Xu, Y., Xu, E., Zhou, M., Wang, B., & Shen, J. (2016). Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells. Oncology Letters, 11, 3054-3060. https://doi.org/10.3892/ol.2016.4338
MLA
Liu, Y., Li, T., Xu, Y., Xu, E., Zhou, M., Wang, B., Shen, J."Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells". Oncology Letters 11.5 (2016): 3054-3060.
Chicago
Liu, Y., Li, T., Xu, Y., Xu, E., Zhou, M., Wang, B., Shen, J."Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells". Oncology Letters 11, no. 5 (2016): 3054-3060. https://doi.org/10.3892/ol.2016.4338