High expression of ENA-78 in lymphatic epithelial cells associated with cholangiocarcinoma promotes lymphangiogenesis / 医学研究生学报

ABSTRACT

ObjectiveLymphatic epithelial cells (LECs) are important links involved in lymphatic metastasis in the microenvironment of cholangiocarcinoma. This study aims to detect the modulation of inflammatory factors and chemokines secreted by LECs after stimulation of cholangiocarcinoma cells, and observe the effects of highly expressed factors on lymphangiogenesis.MethodsThe culture medium of cholangiocarcinoma (RBE, HCCC9810), LECs stimulated by cholangiocarcinoma cell culture medium (CCM), and normal LECs were prepared. Inflammatory factors and chemokines in the culture medium were detected using protein chip. The experiments are divided into the following groups, including a blank control group, CCM group, CCM coupled with Anti-ENA-78 group, Anti-ENA-78 group, ENA-78 group, ENA-78 coupled with SB2252002, and SB225002 group. The relationship between the content of factor and time was investigated using ELISA, while the relation between target factors and lymphangiogenesis obtained by cell proliferation and tubule formation assay.ResultsWe found ENA-78, IP-10, GCP-2, MCP-2, MCP-3, MIP-3a, HCC-1, and Lymphotactin expression increased in LECs supernatant after CCM stimulation. However, I-TAC, MIP-1d, IL-10, MIG, PDGF-BB, and CXCL16 factors showed down-regulation. The secretion of ENA-78 in CCM was relatively low. By ELISA, we found that the ENA-78 protein in RBE-LECs and HCCC9810-LECs gradually increased over time, and reached the plateau phase at the point of 48h. The lymphatic tube forming ability of LECs cultured in CCM was significantly increased compared with that of the control group, and this ability could be partially weakened by ENA-78 neutralizing antibodies. In the exogenous ENA-78 protein group, the lymphatic tube formation ability was as well significantly increased compared with that in the control group, and this ability could be effectively blocked by the IL-8B inhibitor.ConclusionThe increased secretion ENA-78 of lymphatic epithelial cells induced by cholangiocarcinoma may play a role in promoting lymphangiogenesis through the IL-8B receptor.