Effects Of Annexin A4 On EMT And Migration And Invasion In Colorectal Cancer

Background and Objective:Colorectal cancer is a common malignant tumor of digestive tract, and the incidence rate is increasing in recent years.The incidence of colorectal cancer is a complex process involving multiple genes and multiple steps,so its exact pathogenesis is still not clear.Annexins are a superfamily binding with negatively charged phos-pholipid membranes in a Ca2+-dependent manner.Annexins participate in cytoskeletal organization,exocytosis, endocytosis, and apoptosis,they also play important roles in proliferation, invasion, metastasis, and drug resistance of cancer.Annexin A4(Anxa4) is one of the Ca2+-regulated and phospholipid-binding annexin superfamily proteins.Recently, the expression and relocalization of Anxa4 have been reported that it is associated with the development of metastasis in some invasive malignancies suggesting a role for this protein in regulates epithelial cell migration and invasion.However, the specific mechanism is not clear. In this study,we employ si RNA-mediated gene silencing of Annexin A4 to explore the effect of Annexin A4- si RNA on the expression of Annexin A4 and epithelial-mesenchymal transition- related gene and to evaluate its effects on the biological behaviors(proliferation, invasion, and migration) of colorectal carcinoma cells,further enrich the process of molecular mechanism of annexin A4 in colorectal cancer invasion and metastasis.Methods: The expression of Annexin A4 and epithelial-mesenchymal transition- related molecules in three kinds of colorectal cancer cell lines(SW480、SW620、HT-29)were detected by q RT-PCR and western blot.After analysis the correlations between the expression of Annexin A4 and epithelial-mesenchymal transition in colorectal cancer, we selected the cell lines which were suitable for our experimental study.After RNAi was performed in colorectal cancer HT29 cells to downregulate the ecpression of Annexin A4, at first, the expression of Annexin A4 and epithelial-mesenchymal transition- related molecules(such as:E-caderin、N-caderin) were detected by q RT-PCR and western blot, then cell proliferation assay was performed by MTT method and Colony formation test,and then in vitro cell’s invasion ability was detected by transwell cabinet test,at last migration ability of HT29 ceels was detected by scratch test.Results:1. The q RT-PCR results showed that the expression of Annexin A4 in normal colorectal NCM460 cells was very low;By sharp contrast,Annexin A4 were strongly overexpressed in colorectal cancer SW620、SW480、HT29 cells, especially in colorectal cancer HT29 cells2. The Western Blot results revealed the expression of Annexin A4 in colorectal cancer HT29 cells was the highest in SW620,SW480,HT29 cells, which was consistent with the results of q RT-PCR assay.3.The q RT-PCR and Western blot results showed that in colorectal cancer HT29 cells, the E-caderin expression was significantly lower than that in SW620 and SW480 cells, while the N-caderin expression was significantly higher than that of colorectal cancer SW620 and SW480 cells.4.After RNAi was performed in colorectal cancer HT29 cells to downregulate the expression of Annexin A4, the q RT-PCR resuluts showed that the expression of Annexin A4, N-cadherin, MMP9, MMP2,ZEB1, Snail, Slug and Vimentin in HT-29-ANXA4-si RNA cells were decreased(P<0.05) while the expression of E-cadherin were increased(P<0.001).5.After RNAi was performed in colorectal cancer HT29 cells to downregulate the ecpression of Annexin A4, the Western Blot results showed that the protein expression level of E-cadherin in HT29-ANXA4-si RNA cells was significantly higher than that of si NC group while the protein expression level of N-caderin was significantly decreased.6.After RNAi was performed in colorectal cancer HT29 cells to downregulate the ecpression of Annexin A4, the growth speed was slower in HT29-ANXA4-si RNA cells than in si NC cells showed by MTT method as well as Colony formation test.7.After RNAi was performed in colorectal cancer HT29 cells to downregulate the ecpression of Annexin A4,both the in vitro invasive and migrate ability of HT29-ANXA4-si RNA cells detected by transwell cabinet test and scratch testwas was decreased.Conclusion:1.There were close interrelaion between the ecpression of Annexin A4 and EMT- related molecules of colorectal cancer,and Annexin A4 could serve as a potential invasion and migration biomarker for colorectal cancer.2.The downregulation of Annexin A4 in colorectal cancer cells could inhibit the ability of proliferation, metastasis and migration in vitor.