Effect Of Neuropilin-2 Silenced On Proliferation Of Colon Cancer Cell Line HCT-8

Objective To observe the effects of down-regulate the expression of Neuropilin-2(NRP-2)by RNAi technique on the proliferation of HCT-8 colon cancer cells in vitro.Methods Normatively cultured HCT-8 colon cancer cells by to logarithmic phase,the fluorescent siRNA of NRP-2 was transfected into HCT-8 cells by liposome.After 48 hours of common culture,the transfection efficiency of HCT-8 was observed by fluorescence microscope.According to the different transfection materials,colon cancer cells were divided into three groups: transfection group(transfected with siRNA-NRP-2),negative control group(transfected with siRNA-NControl)and blank control group(transfection buffer PBS).Using real-time reverse transcription PCR(RT-qPCR)to detect the expression of NRP-2 mRNA in each group of cells.Western blot was used to detect the expression of NRP-2 protein in different groups of cells.The optical density of each group was measured by CCK-8 assay in order to determine the proliferation of cells.Immunocytochemistry was used to detect the expression of Ki-67 protein in each group of cells.The proliferative capacity of cells in each group was examined by colony-forming unit assay.Acridine orange/propranidine iodide(AO/PI)staining method was used to detect the percentage apoptosis of HCT-8 in each group.Every experiment was repeated more than 3 times in each group.All data were analyzed by SPSS22.0 statistical software.The measurement data were expressed by `x±s.One-way ANOVA was used to compare multiple groups.P<0.05 was considered statistically significant.Results 1 After transfection of fluorescent siRNA48 h by liposome Lip-2000,fluorescence microscope was used to count the cells that were successfully transfected.The transfection efficiency of the experiment was more than 80% in three times.2 Detection the relative expression of NRP-2 mRNA in each group of cells by RT-qPCR.It was significant that transfection group was lower than negative control group and blank control group,and the difference was statistically significant(P< 0.05).3 The expression levels of NRP-2 protein in HCT-8 cells was detected by Western blotting.Compared with negative control group and blank control group,the expression of NRP-2 protein in transfection group was significantly lower than that,and the difference was statistically significant(P<0.05).4 The results of MTT showed that compared with negative control group and blank control group,the optical density of transfection group has a significant difference at 24 h,48h,72 h and 96 h time points,and the difference was statistically significant(P<0.05).5 The results of immunocytochemical staining showed that compared with the negative control group and blank control group,the expression of Ki-67 protein was significantly decreased in the transfection group,and the difference was statistically significant(P < 0.05).6 The results of acridine orange/pyridine iodide staining showed that the apoptosis level of transfected cells was significantly higher than negative control group and blank control group,and the difference was statistically significant(P < 0.05).Conclusion The technique of RNAi can silence the expression of NRP-2,and inhibit the ability of proliferation and promote apoptosis.Figure7;Table7;Reference 125...