Receptor-Interacting Protein Kinase 1 Promotes Cholangiocarcinoma Proliferation And Lymphangiogenesis Through The Activation Protein 1 Pathway

Abstract

Cheng-Zong Li,1,* Yu-Xiang Lin,2,* Tian-Cong Huang,1 Jun-Yong Pan,1 Gao-Xiong Wang1 1Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Fujian Medical University (Donghai District), Quanzhou 36200, People’s Republic of China; 2Department of Surgery Ward 6, The Second Affiliated Hospital of Fujian Medical University (Licheng District), Quanzhou 36200, People’s Republic of China*These authors contributed equally to this workCorrespondence: Gao-Xiong WangDepartment of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital Of Fujian Medical University (Donghai District), 950 Donghai Avenue, Fengze, Quanzhou 36200, People’s Republic of ChinaTel [phone]Fax [phone]Email [email protected]: Receptor-interacting protein kinase 1 (RIPK1) is an important upstream regulator of multiple cell signaling pathways including inflammatory signals. RIPK1 is reported to be closely associated with the prognostic implications of cancer, especially epithelial tumors. But its role in proliferation and lymphangiogenesis in cholangiocarcinoma (CCA) remains unclear and requires further investigation.Patients and methods: Expression of RIPK1 in human CCA tissues and CCA cell lines (QBC939, HUH28 and CCPL-1) was measured using qPCR, immunoblotting and immunohistochemistry. Silencing of RIPK1 was achieved by transduction of CCA cells via lentiviral plasmids (LV3-H1/GFP&Puro) encapsulating RIPK1 shRNA (LV-shRIPK1) or negative control shRNA (LV-shNC), and puromycin was used to select stable colonies. Proliferation and lymphangiogenesis were assessed in vitro by CCK-8 and matrigel-based tube formation assays, respectively. Activity of the activation protein-1 (AP-1) was evaluated by double-luciferase reporter gene assay. Protein expression of JNK, P38MAPK, ERK1/2, AP-1, P-AP-1, E-cadherin, N-cadherin and vascular endothelial growth factor-C (VEGF-C) was measured by immunoblotting or ELISA. An orthotopic CCA model in null mice was generated by transplanting QBC939 LV-shRIPK1, LV-shNC and control cells to further evaluate the role of RIPK1 on lymphangiogenesis in vivo. Immunohistochemistry was utilized to evaluate the expression of RIPK1 and VEGF-C, and tumor lymphatic vessels in the CCA model mice.Results: Upregulated expression of RIPK1 in CCA tissues was closely related to tumor size, lymph node metastasis and poor prognosis. RIPK1 promoted proliferation and lymphangiogenesis in CCA cells, and regulated the activation of JNK and P38MAPK-mediated AP-1/VEGF-C pathway. Finally, in vivo animal experiments in the orthotopic CCA mouse model further confirmed the function of RIPK1 in lymphangiogenesis.Conclusion: This is the first report demonstrating the role of RIPK1 in proliferation and lymphangiogenesis through the MAPK (JNK and P38MAPK)- AP-1 pathway in CCA.Keywords: cholangiocarcinoma, RIPK1, AP-1, VEGF-C, proliferation, lymphangiogenesis

Keywords

• cholangiocarcinoma
• ripk1
• ap-1
• vegf-c
• proliferation
• lymphangiogenesis