Effects Of SiRNA Interference On Slug Gene In IEC-6 Cells On Radiation Sensitivity

Objective Evaluating the effect and mechanism of silencing Slug by RNA interference on the radiosensitivity of intestinal crypt cells(IEC-6)in vitro.To investigate the protective effect of Slug gene on intestinal stem cells,and to find a new strategy to reduce the radiation damage of normal intestinal tissue.Methods(1)Rat intestinal crypt epithelial cells(IEC-6)in vitro were used to experiment.According to the c DNA sequence of Slug gene,3 specific sequences(siRNASlug-1,siRNA-Slug-2,siRNA-Slug-3)and 1 non specific sequences(siRNA-NC)were designed and synthesized.(2)The IEC-6 cells were to be transient transfection by the transfection technology of Ruibo biological company.These cells were transfected with siRNA-Slug(experimental group),siRNA Negative Control(NC-siRNA group)and non transfected small interfering RNA(blank control group).(3)The transfection efficiency was observed under fluorescence microscope,and the optimal transfection concentration and transfection time were selected for the following experiments.(4)Western Blot was used to detect the protein expression of Slug and PUMA in each groups.(5)CCK8 assay was used to observe the growth inhibition of IEC-6 cells under different doses of X-ray.(6)The apoptotic rate of cells were detected by flow cytometry(FCM).(7)The radiosensitivity of IEC-6 cells was observed by colony formation assay.(8)All data were presented as the mean ± SD and determined by one-way analysis of variance(ANOVA).The statistical analyses were performed using the SPSS17.0 or Graph Pad Prism5 software.A value of p < 0.05 was considered statistically significant.Result(1)siRNA-Slug can be successfully transfected into IEC-6 cells by the transfection technology of Ruibo biological company.(2)We successfully selected the best interference effect siRNA-Slug by Western blot.Compared with negative control and blank control groups,the protein expression of Slug in siRNA-Slug group was lower obviously,which treated with X-ray after 24 hours(P<0.01);The protein expression of PUMA in siRNA-Slug cells was significantly higher than that in negative control group and blank control group(P<0.01).With the down-regulation of Slug expression in cells of the experimental group,the expression of PUMA was increased.(3)In the ranges of 0~6Gy,cell proliferation activity decreased with the increase of X-ray dose.But the growth inhibition of experimental group was significantly increased under the 6Gy dose of Xray,compared with negative control and blank control groups,and the difference was statistically significant(P<0.01).(4)In the experimental group,apoptosis rate was increased after X-ray treatment,compared with negative control and blank control groups,and the difference was statistically significant(P<0.01).(5)The clone formation assay showed that the survival rate of the experimental group was significantly lower than that of the negative control and the blank control groups,the difference was statistically significant(P<0.01).Conclusion Silencing Slug gene by siRNA could enhance the sensitivity of IEC-6 cells to X rays,which is realized by inducing the up regulation of PUMA expression.