Molecule Mechanisms Of Laminin Receptor Signal In The Cholangiocarcinoma Cell Invading Endothelial Barrier

Purpose:Metastasis is a complicated progress,including the colonization of selective, secondary organ sites by dissemination of tumor cells from their primary location.Early steps in the metastatic cascade include the shedding of cancer cells from the primary neoplasm,the invasion of extracellular matrices,and the intravasation of tumor cells into lymph and/or blood vessels and so on.Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis.However,little is known about the mechanism by which cancer cells traverse this endothelial barrier.Tumor metastasis has been studied extensively from different aspects.The key point exists in searching a regulative point or a trigger point,which can influence various steps correlative phenotypes during tumor metastasis.It's been proved that laminin receptor(LNR) may plays important roles in mediation of cholangiocarcinoma cell invasion endothelial cell barrier.Therefore,we hypothesize that downregulation of LNR expression on cholangiocarcinoma cell might interfere information transfer between the tumor cell and endothelial cell.There may be a new therapeutic approach to decrease tumor aggressiveness. The experiments outlined in this work were designed to test this hypothesis.Experimental Design:In the first part of the experiment,we examined the expression of LNR in high and low metastatic cholangiocarcinoma cell by FCM.Results showed the expression of LNR in high strain was higher than in the low one.Downregulation of LNR expression on high metastatic cholangiocarcinoma cell was transfected by LNR ASODN.In the second part of the experiment,we established the direct contact and Transwell^? co-culture system of HUVEC and cholangiocarcinoma cell.The morphology change of HUVEC was observed by light microscope and SEM.The functional change of HUVEC was examined by IMF,LSCM,real-time quantitative PCR,western blot analysis and gelatin zymography.In the last part of the experiment,the morphology and functional change of HUVEC was examined when it was co-cultured with high metastatic cholangiocarcinoma cell with transfected into LNR ASODN.And we analyze the laminin receptor signal transmission pathway in the mediation of metastatic process of cholangiocarcinoma cell invasion endothelial barrierResults:1.The expression of LNR was different in high and low metastatic cholangiocarcinoma cells.The high expression of LNR was related with high metastatic potential in cholangiocarcinoma cells.2.LNR expression in high metastatic cholangiocarcinoma cells was downregulated after transfected with LNR ASODN.3.Cholangiocarcinoma cells adhered to HUVEC quickly and induced HUVEC tube formation in vitro.The junction of HUVEC was loosen after cholangiocarcinoma cells adhering to HUVEC.4.The ability of adhesion cholangiocarcinoma cells with HUVEC decreased after the cholangiocarcinoma cells transfected with LNR ASODN.5.The expression of F-actin and pp125FAK in HUVEC increased when they were co-cultured with cholangiocarcinoma cells.LNR ASODN can downregulate these effect.6.The level of mRNA and protein of MMP-2,MMP-9 and uPA increased when they were co-cultured with cholangiocarcinoma cells.LNR ASODN can downregulate these effect.Conclusion:1.LNR is the most important adhesion molecule at the signal between HUVEC and cholangiocarcinoma cells.2.HUVEC was contracted by ways of actin polymerizing and depolymerizing when co-cultured with cholangiocarcinoma cells,pp125FAK is the key of HUVEC and cholangiocarcinoma cells signal pathway.3.pp125FAK-MAPK-ERK signal pathway was activated by cholangiocarcinoma cells in HUVEC.The expression of proteolytic enzyme increased in HUVEC when they co-cultured with cholangiocarcinoma cells.4.LNR paracrine pathway play an important role in the course of invasion the endothelial barrier.