The expression of Par-4 gene in PC12 cells after hypoxia injury and anti-apoptosis effect of Par-4 antisense oligodeoxynucleotide

Objective To investigate the effects of hypoxia on the expression of prostate apoptosis response-4(Par-4) gene in PC12 cells,and anti-apoptosis effect of Par-4 antisense oligodeoxynucleotide(AS-ODN).Methods PC12 cells were cultured in normal or hypoxic condition.Cationic lipid-mediated AS-ODN was transfected into PC12 cells before hypoxia.And mismatch ODN (MS-ODN) was also transfected into the cells as control.Morphological observation and the detection of anti-apoptosis effects of Par-4 AS-ODN on PC12 cells were done with laser scanning confocal microscope by double staining the cells with acridine orange/ethidium bromide(AO/EB). Percentage of apoptosis was evaluated with flow cytometry.The protein eexpression of Par-4 was determined by Western blot.Caspase-3 relative activity was detected by colormetric assay.Results Compared with that of normal control group,the protein level of Par-4 in PC12 cells was significantly increased after hypoxia for 12 hours(157.42±7.53 vs 10.51±3.36,P0.01).Par-4 AS-ODN inhibited the protein expression of Par-4 in PC12 cells after hypoxia 24 hours in a dose-dependent manner.The Par-4 protein in hypoxia group reached 157.42±7.53,and 10μmol/L AS-ODN can significantly down-regulat the Par-4 protein expression(31.79±3.09, P0.01).Par-4 AS-ODN could inhibit the apoptosis of PC12 cells after hypoxia for 24 hours.Transfection of 10μmol/L AS-ODN made apoptotic rate decreased to (34.5±3.5)% from (69.3±5.7)%(P0.05).Par-4 AS-ODN inhibited the increase of Caspase-3 relative activity in PC12 cells exposed to hypoxia for 24 hours(0.549±0.078 vs 0.917±0.051,P0.05).Conclusion Par-4 gene might be involved in the damage of PC12 cells after hypoxia.Par-4 AS-ODN could inhibit the apoptosis of PC12 cells exposed to hypoxia.Its mechanism may be related to the inhibition of activation of Caspase-3.