RANKL Promotes Migration and Invasion of Hepatocellular Carcinoma Cells via NF-κB-Mediated Epithelial-Mesenchymal Transition

<div><p>Background</p><p>Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.</p><p>Methods</p><p>Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability.</p><p>Results</p><p>We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (<i>p</i><0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, <i>in vitro</i> experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells.</p><p>Conclusions</p><p>RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.</p></div

RANKL activated the NF-κB pathway in HCC cells.

<p>HepG2 cells were incubated with 100 ng/ml RANKL for 0, 15, 30 and 60 minutes. A. Western blot analysis indicated that total NF-κB p65 protein was up-regulated significantly upon RANKL treatment in a time-dependent manner. B. Treatment with RANKL did not change the expression of NF-κB p65 in cytoplasm as incubation time prolonged. C. RANKL stimulation significantly increased the expression of NF-κB p65 in the nucleus in a time-dependent manner. D. Western blot analysis revealed that phosphorylation of NF-κB p65 increased in a time-dependent manner upon RANKL treatment. * <i>p</i><0.05 compared with 0 min group.</p

Allelic and genotypic meta-analysis of the KIF1B polymorphism at rs17401966 in all cohorts under alternative genetic models.

<p>HCC, hepatocellular carcinoma; CI, confidence interval; OR, odds ratio.</p

RANKL regulated the expression of EMT-related molecules in HCC cells.

<p>HepG2 and Huh7 cells were incubated with 100 ng/ml RANKL or PBS as control for 24 h. A. Both qRT-PCR and Western blot analyses revealed RANKL stimulation significantly promoted the expression of N-cadherin, Snail and Twist, inhibited E-cadherin, whereas the expression of vimentin and Slug did not show obvious changes. B. After treated with RANKL, Huh-7 showed a similar change in EMT markers as HepG2 cell. ** <i>p</i><0.01 compared with control.</p

Allelic and genotypic meta-analysis of KIF1B polymorphism at rs17401966 in Chinese cohorts under alternative genetic models.

<p>HCC, hepatocellular carcinoma; CI, confidence interval; OR, odds ratio.</p

Forest plot presenting the meta-analysis of KIF1B polymorphisms and the susceptibility to hepatocellular carcinoma under the genotypic model G vs.A in the Chinese subgroups.

<p>The horizontal lines represent 95% confidence intervals for estimating the outcome of the G allele versus the A allele in the meta-analysis. (▪) Overall estimates of the effects.</p

Forest plot presenting the meta-analysis of KIF1B polymorphisms and the susceptibility to hepatocellular carcinoma under the genotypic model G vs.

<p><b>A in the 13 cohorts.</b> The horizontal lines represent 95% confidence intervals for estimating the outcome of the G allele versus the A allele in the meta-analysis. (▪) Overall estimates of the effects.</p

Additional file 1: of FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-β/Smad2/3 signaling pathway

Supplementary Methods. (DOC 37 kb

Additional file 2: of FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-β/Smad2/3 signaling pathway

The additional file contains 6 sub-files. (ZIP 2185 kb