| Objective :To investigate the influence of knocking down EphA2 gene with small interference RNA(siRNA) on biological behavior of human prostate cancer PC3 cell, including cellproliferation, migration force,apoptosis, and the vasculogenic mimicry.So as to provide experimental evidence for the gene targeted therapy of prostate cancer.Methods : PC3 cells(EGFR exon 19 del and sensitive to EGFR-TK1, such as gefitinib) were cultured and according to the conditions of transfection with or without transfection of siRNA,the most effective siRNA was picked through test of Q-PCR and Western blot after24 hours.PC3 cell was divided into three groups: PC3 group, PC3+ NC siRNA group and PC3 + EphA2 siRNA group.Epha2-siRNA were transfected and gifitinib were added into PC3 cells for 48 h, then the apoptosis was measured by FACS; MTT test was used to measure the proliferation of PC3 cell; Transwell test was used to observe invasiveness.At the same time, the PC3 effect on vasculogenic mimicry in three-dimensional culture conditions were observed after transfection.Results:(1).Realtime PCR results showed that compared with the control, negative control had no effect, and the EphA2 expression levels in the groups transfected with siRNA-444?siRNA-733?siRNA-1533?siRNA-2191 reduced 54.60%(P<0.0001)?52.01%(P<0.0001)?93.50%(P<0.0001)?74.23%(P<0.0001),indicating that these siRNA sequences could silence the EphA2 gene. Western Blot results showed that compared with the control,negative control had no effect and the protein expression levels in the groups transfected with siRNA-444?siRNA-733?siRNA-1533 ? siRNA-2191 reduced 46.76%( P<0.001) ? 45.02%(P<0.001)?80.02%(P<0.001)?64.30%(P<0.001), indicating that these siRNA sequences could effectively inhibit the expression of EphA2 gene. The siRNA-1533 was the most effective sequence.(2).Cell proliferation was estimated at 0h, 24 h, 48 h, 72 h after transfection by MTT test. Comparing the survival rate(according to the measured OD values of cells) of three groups of cells, We found,at 48 h, 72 h after transfection, survival rate of PC3+EphA2 siRNA group was different to the others(P<0.05),and the interference effect in 72 h was the most efficient of all(F=51.23,P<0.01).(3).Transwell method was used to measure the invasiveness after transfection. Results showed that: the cell number of PC3 + EphA2 siRNA group going through the Matrigel gel were significantly less than the number of PC3 group and PC3 + NC siRNA group(F=29.00,P<0.01), while the cell number between PC3 group and PC3 + NC siRNA group was no large difference(P>0.05);(4).Flow cytometer was used to measure cell apoptosis at 48 h after transfection. Results showed that: the cell apoptosis rates of PC3 +EphA2 siRNA group, PC3 group and PC3 + NC siRNAgroup was(17.56±1.21)%,(3.98±0.24)%and(4.02±0.28)%, respectively. The apotheosis of PC3+EphA2 siRNA group was high(F=148.14,P<0.01),while the cell apoptosis rate of PC3 group and PC3 + NC siRNA group was no large difference(P>0.05);(5).After transfection of 48 h three cells in three-dimensional culture group, 24 h after termination of training,observation of each tubular structure arrangement and complete degree.Results showed that:the Number of tubular structure of PC3 +EphA2 siRNA group, PC3 group and PC3 + NC siRNAgroup was8.5±2.3,15.4±3.6 and 14.2±1.8, respectively.The apotheosis of PC3+EphA2 siRNA group was low(F=58,P<0.01), while the cell apoptosis rate of PC3 group and PC3 + NC siRNA group was no large difference(P>0.05).Conclusion:(1).EphA2 gene silencing can effectively inhibit the growth of human prostate cancer PC3 cells, invasion of the cell was significantly decreased, induce cell apoptosis and inhibit the vasculogenic mimicry formation of PC3 cell;(2).Targeting EphA2 gene interference technology can provide targeted therapy for prostate cancer. |
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