Study Of FABP6 On Proliferation And Migration Of Colon Cancer Cells And Molecular Mechanism

BackgroundColorectal cancer is one of the most common tumors in the digestive tract.In recent years,the incidence rate in developing countries is getting higher and higher,and the incidence rate below the age of 50 is also getting higher and higher.The high morbidity and mortality rate seriously threaten human health.The pathogenesis of colorectal cancer is complex,from the intestinal environment factors to a variety of gene mutations are involved in the occurrence and development of colorectal cancer,of which,the role of bile acid in the development of colorectal cancer is also increasingly clear,especially the sub-bile Acid,can promote DNA damage in colorectal cancer epithelial cells,and then mutate,eventually leading to cancer.The human ileal fatty acid-binding protein(FABP6)is the only protein in the gut that binds bile acids and is involved in the digestion,absorption,metabolism,and hepatic intestinal circulation of bile acids.It can reduce the toxic effects of bile acids on the intestinal epithelium.However,there are two human fatty acid-binding proteins.The isoforms,type 1 and type 2,have been studied in the past and show that type 2 fatty acid binding protein is highly expressed in colorectal cancer and is involved in the development of colorectal cancer.However,recent studies have shown that high expression of colorectal cancer is type 1 fatty acid-binding protein,which is a typical short subtype with 49 amino acids at the N-terminus,is also known as the long-form subtype.In this article,to distinguish two subtypes of proteins,the long fragment is called FABP6-L.The distribution of the two in the cells,the fluidity of the lipids,and the structure are very different.This study aims to study the roles and molecular mechanisms of two isoforms in colon cancer,with a view to revealing the pathogenesis of colorectal cancer.ObjectivesIn this study,type 2 FABP6 overexpression plasmid and type 1 FABP6 interfering RNA(FABP6-L)were constructed.Colon cancer cells HCT116 and HCT8 were studied.The effects of FABP6 protein on the proliferation and migration of colon cancer cells and the underlying molecular mechanism were analyzed..Methods1.Construction of type 2 FABP6 overexpression plasmid and purchase of type 1 FABP6 interfering RNA,transfection of HCT116 and HCT8 cells,Western blotting assay to detect the expression of type 1 and type 2 FABP6 after transfection.2.The effect of type 1 and type 2 FABP6 protein on proliferation of colon cancer cells was detected by CCK8 assay.3.The effects of type 1 and type 2 FABP6 proteins on the migration of colon cancer cells were examined using cell scratches and Transwell chamber assays.4.Western blotting was used to detect the effect of type 1 and type 2 FABP6 protein on the expression of related proteins in colon cancer cells.Results1.The expression of FABP6 type 2 overexpressing plasmid and type 1 FABP6 interfering RNA were transfected into HCT116 cells and HCT8 cells.The results of Western blotting showed that the expression of type 2 FABP6 was significantly increased in colon cancer cells,and the expression of type 1 FABP6 was significantly reduced in colon cancer cells.2.The results of CCK8 assay showed that the growth rate of cells after transfection of type 2 FABP6 overexpression plasmid in colon cancer HCT116 and HCT8 cells was significantly slower than that of the control group(p<0.05),the difference was statistically significant;in colon cancer HCT116 and HCT8 After transfected with type 1 FABP6 interfering RNA in cells,the growth rate of the cells was significantly slower than that of the control group(p<0.05),the difference was statistically significant.3.The results of cell scratch test showed that after transfection of type 2 FABP6 overexpression plasmid in colon cancer HCT116 and HCT8 cells,the healing rate of cell scratches was significantly slower than that of the control group;in colon cancer HCT116 and HCT8 cells After transfected with type 1 FABP6 interfering RNA,the healing rate of cell scratches was significantly slower than that of the control group.4.Transwell assay results showed that: after transfection of type 2 FABP6 overexpression plasmid in colon cancer HCT116 and HCT8 cells,the number of transmembrane cells in the experimental group was 52.6±16.9 and 73.4±6.1,respectively;and the number of membrane cells in the control group was 192.6±28.6 and 165.1±20.4,respectively.The t values were 11.9 and 12.17,respectively,both p<0.001,the difference was statistically significant;After transfecting type 1 FABP6 interfering RNA in colon cancer HCT116 and HCT8 cells,the number of membrane cells in the experimental group was 28.4±5.8,54.0±9.6,and the number of membrane cells in the control group was 85.3±16.7 and 123.1±17.2,respectively.The t values were 9.1 and 9.9,respectively,all p<0.001,the difference was statistically significant.5.Western blotting results showed that: after transfection of type 2 FABP6 overexpression plasmid in colon cancer HCT116 and HCT8 cells,compared with the control group,the expression of STAT3、p-STAT3、AKT、CyclinD1、p-CyclinD1、N-cadherin were reduced,and the expression of E-cadherin was increased in the experimental group;After transfected with type 1 FABP6 interfering RNA in colon cancer HCT116 and HCT8 cells,compared with the control group,the protein expression of β-catenin 、CyclinD1、p-CyclinD1、Axin2、TCF4、Twist、N-cadherin were reduced,and protein expression ofGSK-3β 、 p-GSK-3β 、 E-cadherin were increased in the experimental group.ConclusionsThe roles and molecular mechanisms of the two FABP6 isoform proteins in colon cancer are different.Type 2 FABP6 protein can inhibit the proliferation and migration of colon cancer cells.The molecular mechanism may be through AKT/STAT3 and EMT signaling pathways;Type 1 FABP6 protein can promote colon cancer cell proliferation and migration,and its molecular mechanism may be through Wnt/β-catenin and EMT signaling pathways.