Effect And Mechanism Of Ampelopsin On Invasion And Metastasis Of Colorectal Cancer By Regulating Dermcidin

ObjectiveThis study selected high-invasive colorectal cancer LoVo cells,and AMP was used to intervene to quantitatively analyze the expression levels of Dermcidin(DCD)genes and proteins.Transwell and scratch experiments revealed that AMP significantly inhibited the ability of cancer cells'migration and invasion.Further studies were focused on exploring the inhibitory effects of ampelopsin(AMP)on the migration and invasion of colorectal cancer by DCD as well as investigating its mechanism.Therefore,the experimental basis for the development of AMP as an effective clinical anti-tumor drug.Development of drugs used for high-efficiency,low-toxicity,anti-tumor invasion and metastasis are highly expected.Methods(?)Expression of DCD in colorectal cancer tissues and cells1.Q-PCR and western-blot were used to detect DCD in normal colorectal tissue and colorectal cancer tissue.The expression of DCD in LoVo,HT-29,SW480,SW620,HCT-116 intestinal cancer cell lines and normal intestinal epithelial HIEC cell lines were detected by Q-PCR and western-blot.2.Construction of LoVo cells that stably expressed pcDNA,WT-DCD,NC-siRNA,DCD-siRNA vectors.Comparison of Q-PCR and western-blot for the successful construction of DCD and the expression of DCD.Transwell chamber migration experiments,cell scratch experiments and Transwell invasion experiments were used to study the migration motility and invasive motility of Control pcDNA,WT-DCD,NC-siRNA,and DCD-siRNA groups.(?)Ampelopsin inhibits the migration and invasion of colorectal cancer cells by DCD1.A series of experiments that recorded at 24,48,and 72 h and different concentrations of AMP(10,20,50,80,100,200,300,500?mol/L)and 5-FU(0.25,5,10,20,50,100?g/mL)by MTT assay to detect the effects of cell proliferation on LoVo cell proliferation.2.The cells were divided into Control group,WT-DCD group,WT-DCD+5-FU group and WT-DCD+AMP group.Except for the Control group used LoVo cells,the other groups were treated with LoVo cells transfected by WT-DCD.The same reason,The cells were divided into NC-siRNA group,DCD-siRNA group,DCD-siRNA+5-FU group and DCD-siRNA+AMP group.The negative Control group was co-stained with NC-siRNA,and the other groups were co-stained with DCD-siRNA in transfected LoVo cells.Transwell chamber migration experiments,cell scratch experiments and transwell invasion experiments were used to study the migration motility and invasive motility of each group cells.To investigate the effects of AMP on the migration and invasion of the corresponding LoVo cells,Q-PCR and western-blot were used to compare AMP expression of mRNA and protein levels in colorectal cancer cells.The effects of AMP on the migration and invasion of the corresponding LoVo cells were investigated.Results(?)The experimental results of DCD in colorectal cancer tissues and cells The results of Q-PCR and western-blot show that the expression of DCD in colorectal cancer tissues are higher than that in adjacent tissues(P<0.01).Q-PCR and western-blot assays show that the expression of DCD in colorectal cancer cells,SW620,LoVo,HT-29,HCT-116,and SW480,are higher than that of normal intestinal cells HIEC(P<0.01),The expression of LoVo cells was the most significant invasive colorectal cancer cells.The results of Q-PCR and western-blot show that the expression of DCD mRNA and protein in WT-DCD group are significantly higher than that in Control,pcDNA,NC-siRNA and DCD-siRNA groups(P<0.01).Based on transwell chamber migration experiments,cell scratch experiments and transwell invasion experiments,the number of migration and invading cells in the WT-DCD group increased compared with Control,pcDNA,NC-siRNA,and DCD-siRNA groups(P<0.01).(?)The experimental results of AMP inhibiting colorectal cancer cells by DCD1.The results of Q-PCR and western-blot showed that the expression levels of DCD mRNA and protein in WT-DCD+5-FU and WT-DCD+AMP groups were significantly lower than those in the WT-DCD group(P<0.01).Based on transwell chamber migration experiments,cell scratch experiments and Transwell invasion experiments,the number of migration and invading cells in the WT-DCD group increased significantly compared with the Control group,(P<0.01).By comparing with the WT-DCD group,the number of migration and invading cells in the WT-DCD+5-FU and WT-DCD+AMP groups were significantly decreased(P<0.01).2.The results of Q-PCR and western-blot show that the expression levels of DCD mRNA and protein in DCD-siRNA+5-FU and DCD-siRNA+AMP groups were significantly lower than those in DCD-siRNA groups(P<0.01).Through transwell chamber migration experiments,cell scratch experiments and transwell invasion experiments,the number of migration and invading cells in DCD-siRNA group almost unchanged compared with NC-siRNA group(P>0.05).Compared with NC-siRNA group,the number of migration and invading cells in DCD-siRNA+5-FU and DCD-siRNA+AMP groups significantly reduced(P<0.01).Compared with the DCD-siRNA group,the number of migration and invading cells in the DCD-siRNA+5-FU and DCD-siRNA+AMP groups were significantly decreased(P<0.01).Conclusion(?)DCD are highly expressed in colorectal cancer tissues.The expression of DCD in colorectal cancer cell lines from SW620,LoVo,HT-29,HCT-116 and SW480 are higher than that of normal intestinal cells HIEC.The expression of LoVo cells with high invasive colorectal cancer is the most significantly.DCD can promote the migration and invasion of LoVo cells.(?)Upregulation and knockdown of DCD expression in colorectal cancer cells,AMP significantly inhibited cell migration and invasion.It is confirmed that AMP significantly inhibited the migration and invasion of LoVo cells transfected with DCD.