METHODS: The RIP1
protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1
shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The
protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover,
VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction,
VEGF-C protein levels were measured by immunoblotting and
enzyme-linked
immunosorbent assay, and
VEGF-C promoter and NF-κB activities were quantified using a dual
luciferase reporter assay. The association of NF-κB with the
VEGF-C promoter was analysed by
chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic
transplantation nude mice model were used to evaluate lymphatic tube-forming and
metastasis ability in GBC cells. The expression of RIP1
protein, TNF-α
protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1
protein with TNF-α
protein and lymphatic vessel density was analysed.
RESULTS: TNF-α dose- and time-dependently increased RIP1
protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated
VEGF-C expression at the
protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and
metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α
protein and the lymphatic vessel density in GBC tissues.
CONCLUSION: