RIP1 regulates TNF-?-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-?B-VEGF-C pathway.

ABSTRACT: Background:Tumor necrosis factor alpha (TNF-?) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-?B)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-? signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-?-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear. Methods:The RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-? as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siI?B? 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-?B alpha (I?B?), p-I?B?, TAK1, NF-?B 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-?B activities were quantified using a dual luciferase reporter assay. The association of NF-?B 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-? protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-? protein and lymphatic vessel density was analysed. Results:TNF-? 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-?-mediated NF-?B activation in GBC cells and can regulate TNF-?-mediated VEGF-C expression at the protein and transcriptional levels through the NF-?B pathway. RIP1 can regulate TNF-?-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-? protein and the lymphatic vessel density in GBC tissues. Conclusion:We conclude that RIP1 regulates TNF-?-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-?B-VEGF-C pathway.

SUBMITTER: Li CZ 

PROVIDER: S-EPMC5962258 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature