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Cited by 163Immune Response and InflammationTGF-β signaling in diseasesHepatocellular Carcinoma Treatment and Prognosis

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Toll-like receptor 4 signaling promotes epithelial-mesenchymal transition in human hepatocellular carcinoma induced by lipopolysaccharide

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Jing, Ying-Ying, Han, Zhi-Peng, Sun, Kai, Zhang, Shan-Shan, Hou, Jing, Liu, Yan, Li, Rong, Gao, Lu, Zhao, Xue, Zhao, Qiu-Dong, Wu, Meng-Chao, Wei, Li-Xin·2012·BMC Medicine
DOI 10.1186/1741-7015-10-98PMC PMC3482562
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Abstract

Background: The endotoxin level in the portal and peripheral veins of hepatocellular carcinoma (HCC) patients is higher and lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to inhibit tumor growth. However, in this study, we found that LPS-induced toll-like receptor 4 (TLR4) signaling was involved in tumor invasion and survival, and the molecular mechanism was investigated, Methods: Four HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasion ability of LPS-induced HCC cells and the epithelia-mesenchymal transition (EMT) in vitro and in vivo. A total of 106 clinical samples from HCC patients were used to evaluate TLR4 expression and analyze its association with clinicopathological characteristics Results: The in vitro and in vivo experiments demonstrated that LPS could significantly enhance the invasive potential and induce EMT in HCC cells with TLR4 dependent. Further studies showed that LPS could directly activate nuclear factor kappa B (NF-κB) signaling through TLR4 in HCC cells. Interestingly, blocking NF-κB signaling significantly inhibited transcription factor Snail expression and thereby inhibited EMT occurrence. High expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients. Conclusions: Our results indicate that TLR4 signaling is required for LPS-induced EMT, tumor cell invasion and metastasis, which provide molecular insights for LPS-related pathogenesis and a basis for developing new strategies against metastasis in HCC.

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Figure 1 Metastasis and invasion of HCC cells lines in response to LPS. (A) The wound healing assay was employed to determine the migration of HepG2, Huh7, Hep3B and SMMC-7721 cells in response to LPS. Cells were monitored every 24 hours for two days to evaluate the rate of migration into the scratched area. (B) Invasiveness of cells was determined using the Transwell assay. Cells were treated with LPS (10 μg/ml) for 48 hours, and then plated in the upper chamber of the Transwell and allowed to grow for 24 hours in serum-free medium, 5% fetal bovine serum was placed in the lower chamber. The number of cells that invaded through the Matrigel was counted in ten fields under the ×20 objective lens, and is shown as the mean ± standard deviation (*P < 0.05, **P < 0.01). (A) and (B) are representative of at least three independent experiments (× 200). (C) Pictures of metastatic liver nodules in nude mice by splenic-vein injection of HepG2 and SMMC-7721 cells untreated or treated with LPS. H & E staining was performed on serial sections of metastatic tumors and normal liver (× 200). The arrows indicate the metastatic tumor on the surface of the liver. (D) The numbers of nodules were quantified on nude mice livers (n = 10 per group). Values for individual mice are shown above the bars (*P < 0.05). (E) Survival rate of nude mice six weeks after splenic vein injection of HepG2 and SMMC-7721 cells untreated or treated with LPS. HCC, hepatocellular carcinoma; LPS, lipopolysaccharide.
Figure 1 Metastasis and invasion of HCC cells lines in response to LPS. (A) The wound healing assay was employed to determine the migration of HepG2, Huh7, Hep3B and SMMC-7721 cells in response to LPS. Cells were monitored every 24 hours for two days to evaluate the rate of migration into the scratched area. (B) Invasiveness of cells was determined using the Transwell assay. Cells were treated with LPS (10 μg/ml) for 48 hours, and then plated in the upper chamber of the Transwell and allowed to grow for 24 hours in serum-free medium, 5% fetal bovine serum was placed in the lower chamber. The number of cells that invaded through the Matrigel was counted in ten fields under the ×20 objective lens, and is shown as the mean ± standard deviation (*P < 0.05, **P < 0.01). (A) and (B) are representative of at least three independent experiments (× 200). (C) Pictures of metastatic liver nodules in nude mice by splenic-vein injection of HepG2 and SMMC-7721 cells untreated or treated with LPS. H & E staining was performed on serial sections of metastatic tumors and normal liver (× 200). The arrows indicate the metastatic tumor on the surface of the liver. (D) The numbers of nodules were quantified on nude mice livers (n = 10 per group). Values for individual mice are shown above the bars (*P < 0.05). (E) Survival rate of nude mice six weeks after splenic vein injection of HepG2 and SMMC-7721 cells untreated or treated with LPS. HCC, hepatocellular carcinoma; LPS, lipopolysaccharide.CC BY · PubMed Central
Figure 2 LPS induces EMT in SMMC-7721 cells but not others. (A) Phase-contrast photographs showing the morphology of HCC cells untreated or treated with LPS (10 μg/ml) for 48 h ours (× 200). (B) qPCR was used to detected changes in expression of EMT genes in HCC cells treated with LPS. Results presented represent mean of triplicate experiments ± SEM (*P < 0.05, **P < 0.01). (C) Immunofluorescent staining of E-cadherin and Vimentin was performed in HepG2 and SMMC-7721 cells that were either untreated or treated with LPS, nuclei were counterstained with DAPI (× 200). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; SEM, standard error of the mean.
Figure 2 LPS induces EMT in SMMC-7721 cells but not others. (A) Phase-contrast photographs showing the morphology of HCC cells untreated or treated with LPS (10 μg/ml) for 48 h ours (× 200). (B) qPCR was used to detected changes in expression of EMT genes in HCC cells treated with LPS. Results presented represent mean of triplicate experiments ± SEM (*P < 0.05, **P < 0.01). (C) Immunofluorescent staining of E-cadherin and Vimentin was performed in HepG2 and SMMC-7721 cells that were either untreated or treated with LPS, nuclei were counterstained with DAPI (× 200). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; SEM, standard error of the mean.CC BY · PubMed Central
Figure 3 TLR4 expression in different HCC cells lines. (A) qPCR was used to detected TLR4 mRNA expression in HepG2, Huh7, Hep3B and SMMC-7721 cells (*P < 0.05). (B) FACS analysis for TLR4 protein in HCC cell lines untreated or treated with LPS (10 μg/ml) for 48 hours. The data shown in A and B are from one representative experiment of three performed. HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; TLR4, toll-like receptor 4.
Figure 3 TLR4 expression in different HCC cells lines. (A) qPCR was used to detected TLR4 mRNA expression in HepG2, Huh7, Hep3B and SMMC-7721 cells (*P < 0.05). (B) FACS analysis for TLR4 protein in HCC cell lines untreated or treated with LPS (10 μg/ml) for 48 hours. The data shown in A and B are from one representative experiment of three performed. HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; TLR4, toll-like receptor 4.CC BY · PubMed Central
Figure 4 Alteration of TLR4 influences LPS-induced metastasis and EMT of HCC cells. (A) and (B) HepG2 cells were transduced with adeno-associated virus to express TLR4 and SMMC-7721 cells were transduced with siRNA to knocked down TLR4, and then the cells were treated with LPS. The migration of pretreated HCC cells was determined by the wound healing assay (A, × 200) and the invasiveness of pretreated HCC cells was determined by the Transwell assay (B, × 200). (C) Expression of EMT genes in pretreated HCC cells was detected by qPCR (normalized to β-actin) Results presented represent the mean of triplicate experiments ± SEM (*P < 0.05, **P < 0.01). (D) Pictures of metastatic liver nodules in nude mice by splenic-vein injection of pretreated HCC cells (× 200). The arrows indicate the metastatic tumor on the surface of the liver (n = 10 per group). (E) and (F) The number of nodules was quantified on nude mice livers by splenic-vein injection of pretreated HCC cells. Values for individual mice are shown above the bars (*P < 0.05). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; SEM, standard error of the mean; TLR4, toll-like receptor 4.
Figure 4 Alteration of TLR4 influences LPS-induced metastasis and EMT of HCC cells. (A) and (B) HepG2 cells were transduced with adeno-associated virus to express TLR4 and SMMC-7721 cells were transduced with siRNA to knocked down TLR4, and then the cells were treated with LPS. The migration of pretreated HCC cells was determined by the wound healing assay (A, × 200) and the invasiveness of pretreated HCC cells was determined by the Transwell assay (B, × 200). (C) Expression of EMT genes in pretreated HCC cells was detected by qPCR (normalized to β-actin) Results presented represent the mean of triplicate experiments ± SEM (*P < 0.05, **P < 0.01). (D) Pictures of metastatic liver nodules in nude mice by splenic-vein injection of pretreated HCC cells (× 200). The arrows indicate the metastatic tumor on the surface of the liver (n = 10 per group). (E) and (F) The number of nodules was quantified on nude mice livers by splenic-vein injection of pretreated HCC cells. Values for individual mice are shown above the bars (*P < 0.05). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; SEM, standard error of the mean; TLR4, toll-like receptor 4.CC BY · PubMed Central
Figure 5 LPS/TLR4 induces cells EMT through activation of NF-κB. (A) A dual luciferase assay was performed 48 hours after adding LPS. Relative NF-kB luciferase activity, normalized to Renilla luciferase activity, was expressed relative to that of control, set at 1.0. LPS resulted in a significant increase of luciferase activity in SMMC-7721 cells, but had no effect in HepG2 cells; NF-κB inhibitors suppress NF-κB activation in SMMC-7721 cells (*P < 0.05). (B) Immunofluoresence was used to evaluate the NF-κB p65 binding activity of pretreated HepG2 and SMMC-7721 cells. The p65 subunit of NF-κB in the cytosol was stained green and nuclei were stained red with PI (× 400). (C) and (D) qPCR and western-blot were used to detect Snail expression in pretreated HepG2 and SMMC-7721 cells. The results showed that LPS upregulated Snail expression in SMMC-7721 cells, but not in HepG2 cells, NF-κB inhibitors suppress Snail expression in SMMC-7721 cells (* P < 0.05). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; NF-κB nuclear factor kappa B; TLR4, toll-like receptor 4.
Figure 5 LPS/TLR4 induces cells EMT through activation of NF-κB. (A) A dual luciferase assay was performed 48 hours after adding LPS. Relative NF-kB luciferase activity, normalized to Renilla luciferase activity, was expressed relative to that of control, set at 1.0. LPS resulted in a significant increase of luciferase activity in SMMC-7721 cells, but had no effect in HepG2 cells; NF-κB inhibitors suppress NF-κB activation in SMMC-7721 cells (*P < 0.05). (B) Immunofluoresence was used to evaluate the NF-κB p65 binding activity of pretreated HepG2 and SMMC-7721 cells. The p65 subunit of NF-κB in the cytosol was stained green and nuclei were stained red with PI (× 400). (C) and (D) qPCR and western-blot were used to detect Snail expression in pretreated HepG2 and SMMC-7721 cells. The results showed that LPS upregulated Snail expression in SMMC-7721 cells, but not in HepG2 cells, NF-κB inhibitors suppress Snail expression in SMMC-7721 cells (* P < 0.05). EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; NF-κB nuclear factor kappa B; TLR4, toll-like receptor 4.CC BY · PubMed Central
Figure 6 Immunohistochemistry of TLR4 expression in HCC tissues and its prognostic implication. (A) and (B) The membranous expression of TLR4 was seen to be low in 42/106 of HCC tissues (A), and high in 64/106 (B). Original magnification left × 200, right × 400. (C) Estimated cancer-free survival according to the expression of TLR4 in 106 cases of HCCs (the Kaplan-Meier method). Log-rank test shows that HCC patients in the high TLR4 expression group have poorer cancer-free survival than those in the low TLR4 expression group (P < 0.001). (D) Overall survival was analyzed in the same cohort of HCC patients and the results showed that HCC patients in the high TLR4 expression group also have poorer overall survival than those in the low TLR4 expression group (P < 0.001). HCC, hepatocellular carcinoma; TLR4, toll-like receptor 4.
Figure 6 Immunohistochemistry of TLR4 expression in HCC tissues and its prognostic implication. (A) and (B) The membranous expression of TLR4 was seen to be low in 42/106 of HCC tissues (A), and high in 64/106 (B). Original magnification left × 200, right × 400. (C) Estimated cancer-free survival according to the expression of TLR4 in 106 cases of HCCs (the Kaplan-Meier method). Log-rank test shows that HCC patients in the high TLR4 expression group have poorer cancer-free survival than those in the low TLR4 expression group (P < 0.001). (D) Overall survival was analyzed in the same cohort of HCC patients and the results showed that HCC patients in the high TLR4 expression group also have poorer overall survival than those in the low TLR4 expression group (P < 0.001). HCC, hepatocellular carcinoma; TLR4, toll-like receptor 4.CC BY · PubMed Central

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Authors
Jing, Y.-Y., Han, Z.-P., Sun, K., Zhang, S.-S., Hou, J., Liu, Y., Li, R., Gao, L., Zhao, X., Zhao, Q.-D., Wu, M.-C., & Wei, L.-X.
Affiliations
Second Military Medical University (CN)
ORCID
Venue
BMC Medicine
Publisher
Springer Science and Business Media LLC
Published
2012-08-31
Volume
10
Issue
1
Pages
98-98
ISSN
1741-7015 (ISSN-L)
DOI
10.1186/1741-7015-10-98
PMC
PMC3482562
OpenAlex
W1970916626
Cited by
162
References
43
Citations by year
2026: 8 · 2025: 15 · 2024: 11 · 2023: 13 · 2022: 14 · 2021: 18 · 2020: 9 · 2019: 11 · 2018: 11 · 2017: 13 · 2016: 10 · 2015: 11 · 2014: 11 · 2013: 7
FWCI
3.99
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Medicine (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Natural Science Foundation of China (30921006, 81030041, 2008ZX10002-019, 81000970, SCMRC1102, 2011CB966200, 81101622, 31171321, 30901722)
  • Science and Technology Commission of Shanghai Municipality (10411963100, 10ZR1439900, 11ZR1449500, 10ZR1439600)
  • Science Fund for Creative Research Groups (30921006)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
  • Health Sciences › Medicine › Hepatology › Hepatocellular Carcinoma Treatment and Prognosis
Keywords
TLR4, hepatocellular carcinoma, epithelial-mesenchymal transition, lipopolysaccharide, NF-κB signaling, Snail transcription factor, tumor invasion, metastasis, endotoxin, tumor-free survival, overall survival
MeSH terms
Adolescent, Adult, Aged, Animals, Child, Endotoxins, Female, Carcinoma, Hepatocellular, Humans, Lipopolysaccharides, Male, Mice, Nude, Middle Aged, Signal Transduction, Gene Expression Profiling, Toll-Like Receptor 4, Mice, Young Adult, Epithelial-Mesenchymal Transition

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