The Mechanism Of LOXL1 Promoting Proliferation,Metastasis And Angiogenesis In Intrahepatic Cholangiocarcinoma

Objective Intrahepatic cholangiocarcinoma(ICC)is one subtype of cholangiocarcinoma which is located proximal to the secondary branches of left and right hepatic ducts.It is aggressive and rapidly progressive,usually leading to poor prognosis.Advances in molecular biology offer more possibilities for the diagnosis and treatment of ICC.LOXL1,as a member of the LOX proteins family,has been reported as a pro-carcinogenetic factor in various cancers,but its role in ICC remains to be further studied.Deeper research of the specific role and molecular mechanism of LOXL1 provides a theoretical reference for the search of therapeutic targets in ICC.Methods The expression of LOXL1 was detected in the serum of 5 normal people and 5 ICC patients and 17 pairs of tumor and para-tumor tissue samples.We assessed proliferation,metastasis,and ability of pro-angiogenesis of intrahepatic cholangiocarcinoma by CCK-8,Transwell assay,Colony formation,and Tube Formation assays after knockdown and overexpression of LOXL1 in the intrahepatic cholangiocarcinoma cell lines RBE and 9810,and then detected changes of epithelialmesenchymal transition(EMT),MAPK,and FAK pathway-related proteins.The subcutaneous xenograft model in nude mice was further demonstrated effects of LOXL1 on proliferation and angiogenesis of intrahepatic cholangiocarcinoma in vivo.FBLN5,protein thought to interact with LOXL1,was screened by String database prediction and literature investigation results,and the interaction between the two was proved by Co-Immunoprecipitation(Co-IP)assay.We further confirmed their connection by immunofluorescence in clinical tumor tissues.Lastly,the role of FBLN5 and downstream Integrin ?v?3 in the pro-angiogenic formation of LOXL1 protein was clarified by constructing a mutant type(RGE)of plasmid compared with the wild type RGD domain of FBLN5.Results LOXL1 is highly expressed in the serum of patients with intrahepatic cholangiocarcinoma.Similarly,the expression of LOXL1 in tumor tissues of ICC is also significantly higher than that in adjacent tissues.Further research results showed that LOXL1 can promote the proliferation,metastasis and pro-angiogenesis of intrahepatic cholangiocarcinoma cells in vitro and in vivo,and promote the epithelialmesenchymal transition of intrahepatic cholangiocarcinoma cells.The interaction between LOXL1 and FBLN5 was confirmed by co-immunoprecipitation assay.The immunofluorescence assay shows the same conclusion as well.What is more,the binding of RGD domain on the FBLN5 protein and integrin ?v?3 on the vascular endothelial cell membrane is necessary for LOXL1 to promote angiogenesis in ICC,which can activate MAPK and FAK signaling pathways in vascular endothelial cells.Conclusions LOXL1 promotes the proliferation and metastasis of intrahepatic cholangiocarcinoma cells by regulating EMT and MAPK pathway,and promotes angiogenesis by binding to FBLN5,which relies on the RGD domain of FBLN5 to bind to Integrin ?v?3.The finding provides new choice of molecular biological treatment for ICC patients.