RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway
Authors Li C, Jiang X, Lin B, Hong H, Zhu S, Jiang L, Wang X, Tang N, She F, Chen Y
Received 6 December 2017
Accepted for publication 9 March 2018
Published 16 May 2018 Volume 2018:11 Pages 2875—2890
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Andrew Yee
Peer reviewer comments 2
Editor who approved publication: Dr Ingrid Espinoza
Cheng-Zong Li,1–3,* Xiao-Jie Jiang,1,2,* Bin Lin,1,2 Hai-Jie Hong,1,2 Si-Yuan Zhu,1,2 Lei Jiang,1,2 Xiao-Qian Wang,1 Nan-Hong Tang,1 Fei-Fei She,2 Yan-Ling Chen1,2
1Department of Hepatobiliary Surgery and Fujian Institute of Hepatobiliary Surgery, Fujian Medical University Union Hospital, Fuzhou, People’s Republic of China; 2Key Laboratory of the Ministry of Education for Gastrointestinal Cancer and Key Laboratory of Tumour Microbiology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, People’s Republic of China; 3Department of General Surgery, The Second Affiliated Hospital Of Fujian Medical University, Quanzhou, People’s Republic of China
*These authors contributed equally to this work
Background: Tumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-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-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: We conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway.
Keywords: gallbladder cancer, RIP1, TNF-α, NF-κB, VEGF-C, lymphangiogenesis