| Objective Successfully silenced Neuropilin-1(NRP-1)in colon cancer SW480 cells,the effect of colon cancer epithelial-mesenchymal transition(EMT)induced by TGF-?1 and colon cancer proliferation and migration.Methods Normatively cultured SW480 colon cancer cells until to the logarithmic phase,the cells were treated as follows:1 After SW480 cells were stimulated with concentrations of Ong/mL,2ng/mL,5ng/mL,and 10ng/mL TGF-?1 to induce the EMT for 24h and 48h,the proliferation of cells was measured by CCK-8 experiment.Western blot detected the protein expression change of E-cadherin and Vimentin.2 Transfected siRNA into SW480 cells with lip2000.Colon cancer cells were divided into three groups according to different transfection materials:Blank control group,nonsense sequence group(transfected siRNA-NControl)and transfection group(transfected siRNA-NRP-1).After 48 hours of routine culture,using real-time quantitative PCR(RT-qPCR)and Western blot to detect the expression of NRP-1 mRNA and protein in each group of cells.3 Using the above-mentioned successful methods of induction and transfection,the cells were divided into 4 groups:nonsense sequence group(transfection siRNA-NControl),transfection group(transfected with siRNA-NRP-1),induction group(TGF-?1),transfection+induction group(TGF-?1+transfection siRNA).Western blot was used to detect the expression of E-cadherin and Vimentin;each group was measured by CCK-8 and wound healing assay to determine the proliferation and migration of cells.Results 1 SW480 cells developed obvious EMT after being stimulated with 10ng/mL TGF-?1 for 48h.The cell proliferation was more obvious and the expression of E-cadherin protein was significantly lower than the other three groups.The expression of Vimentin protein was significantly higher than the other three groups.The difference was statistically significant(P<0.05).2 After transfected siRNA with lip2000 for 48 hours,RT-qPCR and Western blot were used to detect the mRNA and protein expression of NRP-1 in each group of cells.The results showed that the expression of NRP-1 in the transfection group was significantly lower than that in the nonsense sequence group and the blank control group(P<0.05).3 Western Blot detection of nonsense sequence group,transfection group,induction group,transfection+induction group showed that the expression level of E-cadherin protein in transfection+induction group is higher than that in induction group,The protein expression of Vimentin in the transfection+induction group was lower than that in the induction group,and the difference were statistically significant(P<0.05).4 The CCK-8 results showed that the OD value of the transfection group was lower than that of the nonsense sequence group,and that of the transfection+induction group was lower than the induction group,and the differences were statistically significant(P<0.05).5 Wound healing assay was used to detect the migration of cells in each group.The migration ability of the transfection group was slower than that of the nonsense sequence group.Similarly,the transfection+induction group was slower than the induction group,and the difference was statistically significant(P<0.05).Conclusion Colon cancer SW480 cells could be induced EMT by TGF-?1,but after successfully silenced NRP-1,they were able to reverse the occurrence of EMT in cells,with reduced cell proliferation and reduced migration ability.Figure 7;Table 8;Reference 180... |
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