A Study Of Survivin Gene To Promote Apoptosis Of HL-60 Cells And Its Effect On The Sensitivity Of Ara-C To The Cells

Objective:To construct a siRNA plasmid expression vector against Survivin and transfected into HL-60 cells with Lipofectamine 2000TM and to explore the effects of shRNA plasmid expression vector on apoptosis and the sensitivity of Ara-C on leukemia.Methods:(1) To construct eukaryotic expression vectors pshRNA-Survivinl (positive), pshRNA-Survivin2 (negative) by semi-quantitative RT-PCR and directional cloning technology.(2) Six groups were classified:HL-60 cells without transfection (blank group), HL-60 cells with Ara-C, HL-60 cells with pshRNA-Survivinl (positive), HL-60 cells with pshRNA-Survivin2 (negative), HL-60 cells with pshRNA-Survivin 1 (positive) and Ara-C, HL-60 cells with pshRNA-Survivin2 (negative) and Ara-C.(3) Vectors were extracted by Endofree Plasmid Extraction Kit and then transfected into HL-60 cells with Lipofectamine 2000TM.(4) The expression of Survivin mRNA was detected by semi-quantitative RT-PCR and the apoptosis was detected by FCM after transtected for 48h.(5) SPSS 11.0 statistical software was used to analyse the statistical data. (?)±S was used to compare these data, P value less than 0.05 was considered statistically significant. Results:(1) The recombinant plasmid pshRNA-Survivin1(positive) and pshRNA-Survivin2 (negative) were constructed successfully, the inserted sequence ShRNA-Survivin was exactly correct by the sequence analysis. (2) The results of the expression of Survivin mRNA which was detected by semi-quantitative RT-PCR were:The relative express quantity of Survivin mRNA of the six groups of HL-60 cells without transfection (blank group), HL-60 cells which Ara-C was added, HL-60 cells with pshRNA-Survivin1(positive), HL-60 cells with pshRNA-Survivin2 (negative), HL-60 cells with pshRNA-Survivin1(positive) and Ara-C, HL-60 cells with pshRNA-Survivin2 (negative) and Ara-C were 0.83±0.01, 0.80±0.02,0.35±0.02,0.77±0.01,0.17±0.01 and 0.75±0.02, respectively. Compared with the blank group, the Survivin mRNA in HL-60 cells with Ara-C, HL-60 cells with pshRNA-Survivin1(positive) group was decreased by 57.8%, the Survivin mRNA in HL-60 cells with pshRNA-Survivin1(positive) and Ara-C group was reduced by79.5%.The results demonstrate that siRNA targeting Survivin mRNA dramasticly inhibit and increase the sensitivity to Ara-C (P< 0.05).(3) With Flow Cytometry, it showed that the index of apoptosis in six groups were:HL-60 cells without transfection (control group):almost no apoptosis,HL-60 cells with Ara-C:5.09±0.30%, HL-60 cells with pshRNA-Survivinl (positive):5.87±0.38%, HL-60 cells with pshRNA-Survivin2(negative) 2.78±0.30%, HL-60 cells with pshRNA-Survivin1(positive) and Ara-C:12.4±0.38%, HL-60 cells with pshRNA-Survivin2(negative) and Ara-C:8.80±0.32%(P< 0.05).Conclusions:(1) The siRNA eukaryotic expression vector against Survivin gene has been successfully conctructed.(2) The expression level of Survivin mRNA in HL-60 cells was inhibited after the siRNA plasmid expression vector against Survivin was transfected into HL-60 cells.(3) RNA interference can increase the sensitivity of Ara-C to HL-60 cells and enhance the apoptosis index of HL-60 cells.(4) It may also be an effective way using RNA interference technology to inhibit Survivn gene in the gene targeting therapy of leukemia.