Preliminary Study On The Effects And Mechanisms Of Fatty Acid-binding Protein 4 On Cholangiocarcinoma Energy Metabolism And Tumor Metastasis

Objective: In order to explore the effects and mechanisms of FABP4 on the adipose-regulated metastasis of cholangiocarcinoma.Methods: Adipose tissues were obtained from mice and breast cancer patients under sterile conditions, and the adipose tissue extract was co-cultured with human cholangiocarcinoma cell lines RBE and Hcccp810 respectively. The glycerol and fatty acid assay kits were used to detect the content of glycerol and fatty acid inculture medium before and after the adipose/cancer cell co-culture. Migration and invasion of cholangiocarcinoma cells were determined by wound healing assay and transwell assays respectively. The expression of intracellular expression levels of MMP2、MMP9 and TGF-β1 after co-culture was detected by western blot assay. The specific fluorescent dye Bodipy was used to determine the effect of co-culture on adipose accumulation in vitro and the effect of FABP4 during this process. The expression levels of EMT related protein were detected by western blot assay in adipose co-cultured cancer cells at different expression pattern of FABP4.Moreover, human cholangiocarcinoma cell line RBE was infected with FABP4 adenoviral vector. The nude mice tumor metastasis model was constructed by injecting cancer cells via the tail vein. RBE cells colonization in the nude mouse organs were visualized using the in vivo imaging system. Hematoxylin-eosin staining assay was used to detect the formation of solid tumors in mice and Ki67 expression level in liver and lung were deceted by immunohistochemistry assay.Results: Both of mouse and human adipose tissue could promote the migration and invasion of human cholangiocarcinoma cells in vitro. When compared with control cells, the content of fatty acid and glycerol of adipose tissue extract was increased( P<0.05), the content of fatty acid in the medium was decreased significantly(P<0.05) while the content of glycerol was almost unchanged after tumor cells co-cultured with adipose tissue. The expression of MMP-9 and TGF-β1 increased in cancer cells co-cultured with adipose. Meanwhile, the expression of MMP-9, MMP-2 was increased. The intracellular adipose accumulation was increased after cells co-cultured with adipose. FABP4 gene could promote not only the intracellular adipose accumulation, but also the migration and invasion of cancer cells. Decreased claudin1, occludin, E-cadherin, β-catenin and increased SNAIL were detected in co-culture cells, which implied the onset of EMT. However, no significant changes were detected of smad3 expression. It suggested that tight junctions between tumor cells might be weaken and EMT might be occurred. Suppression of FABP4 gene function led to the suppression of EMT, while overexpression of FABP4 can promote EMT. Twenty-four hours after tail vein injection, in vivo imaging system showed that adipose can promote cholangiocarcinoma cells metastasis. The fluorescence intensity of liver and kidney of co-cultured group was 28.8% and 11.5% higher than the control group(P <0.05) respectively. FABP4 could promote the migration and invasion of cells in vivo. And the fluorescence intensity of liver, heart and kidney was 26.4%, 7.3% and 22.3% higher than the NC group(P <0.05) respectively. Thirty days after tail vein injection, solid tumor were formed in the liver of mice in adipose co-cultured group detected by HE staining. The expression of Ki67 was higher in adipose co-culture and FABP4 overexpression group, but the expression of FABP4 was not detected in those tissues.Conclusions: The intracellular accumulation of adipose increased the invasion, migration of cholangiocarcinoma cells. Overexpression of FABP4 gene could promote migration, invasion and accumulation of adipose of cancer cells, which at least through the mechanism of EMT protein regulation.