The Effects Of SiRNA Targeting Inhibited FOXO1 Gene Within Different Concentrations Of Glucose On Proliferation And Apoptosis Of Islet Beta Cell Line NIT-1

Objective To evaluate the effect of small interferring RNA(siRNA) targeting FoxO1 gene on the proliferation and apoptosis of NIT-1cells and to investigate the possible pathogenesis, providing theoretical basis for diabetes'gene diagnosis.Methods 1. Screened effective siRNA clips and optimized transfection conditions. Four pairs of FoxO1siRNA(FoxO1siRNA-1﹑FoxO1siRNA -2﹑FoxO1siRNA -3﹑FoxO1siRNA -4)and one pair of negative control siRNA were synthesized. Optimized transfection conditions with FAM labeled negative control siRNA. Four pairs of FoxO1siRNA were transfected into NIT-1 cells by LipofectimineTM2000 according to the optimal transfection conditions. Real-time PCR detected the interference effect of FOXO1 gene after transfected for 24h, 48h, 72h, 96h. Selected the best clips of FOXO1siRNA.and best silent time .2. The islet beta cell line NIT-1 was randomly assigned to each treatment group: 5.6 group (containing glucose 5.6mmol / L), 11.1 group (11.1 mmol / L), and 27.6 group (27.6mmol / L) after culturing for 24 hours. Each glucose concentration group was divided into experiment group (FoxO1siRNA group), Negative control group (Negative controlsiRNA group), No-load liposomes group and Blank control group according to the effective siRNA and the optimal transfection conditions. Transfectied each group, FoxO1 and its target gene expression levels were assessed by Real-time PCR.MTT assay was used to assess the proliferation of the cells.Fluorescence-activated cell sorter analysis was used to determine apoptosis of the cells on the best quiet time.Results (1) After transfecting with FAM labeled negative control siRNA for six hours, observed the intracellular fluorescent color under the fluorescence microscope. It showed: The transfection efficiency and cell viability were high when the concentration of siRNA was 50nM,and siRNA: LipofectamineTM 2000 (v:v) 1:1. The express inhibition rate of FoxO1siRNA-1﹑FoxO1siRNA -2﹑FoxO1siRNA -3﹑FoxO1siRNA -4 all were high after transfected for 72h. The express inhibition rate of FoxO1siRNA -4 was the highest.So choosed the time of transfecting for 72h as the follow-up experiment's detection time. And choosed the FOXO1siRNA-4 as the follow-up experiment's siRNA fragment.(2) Real-time quantitative PCR results showed that: Under the same glucose concentration, The FoxO1mRNA expression level of NIT-1 cells in experimental group was lowest, significantly lower than the negative control siRNA group, empty liposome group and blank group (p<0.05). Different concentrations of glucose group, FoxO1 mRNA expression in each group showed the same trend, that with the increase of glucose concentration (p <0.05).(3) MTT detection cell proliferation showed that: Under the glucose concentration, The proliferation rate of NIT-1 cells in experimental group increased , higher than the negative control siRNA group ,empty liposome group and blank group (p <0.05); The proliferation rate with negative control siRNA group, empty liposome group and blank group showed no significant difference (p> 0.05). Different concentrations of glucose group, the proliferation rate of 27.6 group was significantly reduced (p <0.05). The proliferation ratethe between 5.6 group and 11.1 group showed no significant differences.(4) Flow cytometry detecting the apoptosis of cells showed: Under the same glucose concentration, the apoptosis of NIT-1 cells in experimental group decreased, lower than the negative control siRNA group , empty liposome group and blank group (p <0.05). Different concentrations of glucose group,each sugar concentration cell apoptosis showed the same trend, that is, with the sugar concentration increased, the cell apoptosis.Conclusion (1)LipofectamineTM2000 transfected FOXO1siRNA into NIT-1 cells can specifically, efficiently inhibit FOXO1 gene expression of NIT-1 cells, It is covinient and has high tranfection efficiency. (2)The technology of RNAi knockdown FOXO1mRNA's expression can promote the proliferation and inhibit apoptosis of NIT-1 cells. FOXO1 can inhibit the proliferation and promote cell apoptosis.