The Roles Of DJ-1 On Proliferation And Apoptosis In The Ishikawa Endometrial Carcinoma Cell Line

Objective:The aim of this study was to investigate the silencing effect of the short hairpin RNA (short hairpin RNA, shRNA) recombinant plasmid vector targeting DJ-1 gene and the roles of DJ-1 in cell proliferation and apoptosis in endometrial cancer Ishikawa cells.Methods:1. Design and synthesis three of targeted DJ-1 shRNA, cloned into plasmid pGPU6/GFP/Neo to construct a recombinant plasmid vector, and evaluate the recombinant plasmid vector by enzyme digestion and sequencing. To detect silence action of pGPU6/GFP/neo-DJ-1-shRNA on the DJ-1 gene in endometrial cell lines, the cells were treated for 72 hours as follows:the pGPU6/GFP/neo-DJ-1-shRNA1 group (20 μg/mL), the pGPU6/GFP/neo-DJ-1-shRNAl group (20 μg/mL), the pGPU-6/GFP/neo-DJ-1-shRNA2 group (20μg/mL), the pGPU6/GFP/neo-DJ-1-shRNA3 gr-oup (20 μg/mL), nonspecific interference plasmid control group (20 μg/mL), and cell control group.2.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumassay was used to inve-stigate the cell proliferation after transfection. Ishikawa cellswere treated with various concentrations(2.5μg/mL,5μg/mL, 10μg/mL and 20μg/mL) of pGPU6/GFP-/neo-DJ-1-shRNAl for various times(24h,48h,72h and 96h).3. To further investigate whether the inhibition of DJ-1 induced apoptosis of Ishikawa cells after transfection, we used annexin V and PI to quantitatively determine the type of cell death induced by down-regulation of DJ-1 in Ishikawa cells, especially apoptosis. The cells were treated for 72 hours as follows:the pGPU-6/GFP/neo-DJ-1-shRNA1 group (20 μg/mL), cell control group, empty plasmid control group (20 μg/mL), nonspecific interference plasmid control group (20 μg/mL), and the liposome control group.Results:1. Confirmed by sequencing, three of DJ-1 targeting pGPU6/GFP/Neo-DJ-1-sh-RNA were successfully constructed and were named pGPU6/GFP/Neo-DJ-1-shRNAl, pGPU6/GFP/Neo-DJ-1-shRNA2 and pGPU6/GFP/Neo-DJ-1-shRNA3.2. Real-time PCR was used to detect the DJ-1 mRNA expression after transfe-ction of pGPU6/GFP/neo-DJ-1-shRNA (20 μg/mL) for 72 hours. The level of DJ-1 mRNA in the pGPU6/GFP/neo-DJ-1-shRNA groups was significantly lower than that the control groups(P< 0.01), whereas the control groups had no sense of statistics compared to each other.3. Western blotting was used to detect the DJ-1 protein expression after transfe-ction of pGPU6/GFP/neo-DJ-1-shRNA(20 μg/mL) for 72 hours. The similar result of western blotting analysis was shown. The level of DJ-1 protein in the pGPU6/GFP/neo-DJ-1-shRNA groups was significantly lower than that the control groups(P< 0.01), whereas the control groups had no sense of statistics compared to each other. Overall, after transfecting the DJ-1 interference plasmid into Ishikawa cells, DJ-1 expression was reduced notably.4. The results revealed that pGPU6/GFP/neo-DJ-1-shRNAl inhibited Ishikawa cells proliferation in a dose- and time-dependent manner, the inhibitory rate of cell viability(%) as follows:22.80±0.75,37.50±1.15,43.75±2.74,53.25±4.46; 28.20 ±1.32,43.15±2.06,49.15±4.15,66.57±2.08; 39.70±1.43,52.75±3.53,55.05± 3.22,75.65±3.42; 42.35±1.24,54.00±4.17,65.55±2.63,89.50±4.43.5. To further investigate whether the inhibition of DJ-1 induced apoptosis of Ishikawa cells after transfection for 72 h, we used annexin V and PI to quantitatively determine the type of cell death induced by down-regulation of DJ-1 in Ishikawa cells, especially apoptosis. Flowcytometry analysis showed that early apoptosis rate in the pGPU6/GFP/neo-DJ-1-shRNA1 group (56.78%±0.28%) was obviously higher than that of the cell control group (0.50%±0.03%), the empty plasmid control group (0.15% ± 0.05%), the nonspecific interference plasmid control group (2.52% ±0.04%), and the liposome control group (5.37%±0.22%); necrotic cells and late apoptosis rate in the pGPU6/GFP/neo-DJ-1-shRNA group (32.51%±0.51%) was obviously higher than that of the cell control group (0.47%±0.03%), the empty plasmid control group (0.03%±0.02%), the nonspecific interference plasmid control group (1.63%±0.11%), and the liposome control group (5.02± 0.06%). The differences were statistically significant (P< 0.01).Conclusion:Knocking down DJ-1 promoted the apoptosis and inhibited the proliferation of Ishikawa cells.