Mechanisms And Effects Of Omi/HtrA2 ShRNA On The Apoptosis Of Rat Renal Tubular Epithelial Cells

Objective The reconstructed plasmids which contained green fluorescent protein gene were transfected into NRK-52E by siPORT XP-1.The transfected cells were resisitant to G418(300μg/ml).The expression of Omi/HtrA2 in transfected cells was inhibited.Then to investigate the mechanisms and effects of Omi/HtrA2 short hairpin(shRNA) on the apoptosis of NRK-52E from hypoxia-reoxygenation injury.Methods The cells were indivded into 5 groups:normal groups(n=6),cells were cultured under routine condition;model groups(n=6),hypoxia-reoxygenation injury was performed;HK groups(n=6),transfected with Pgenesil-1/HK;shRNA1 groups,transfected with Pgenesil-1/ Omi/HtrA2 shRNA1;shRNA2 groups(n=6),transfected with Pgenesil-1/Omi/HtrA2 shRNA2. The cells were incubated in 6 well plates;when cells were 50%-60%confluence,the transfection was performed.The medium was refreshed after the first 8 hours with medium DMEM/F12 supplemented with 15%fetal bovine serum(FBS).The second day,the cells were incubated with medium containing G418(300μg/ml).The fluorescent microscopy was to examine the expression of fluorescent protein.After two weeks,there were only transfected monoclone cells in the culture plates.Excepting normal group,the other four groups were cultured with medium oxygen-free.The cells were exposed to gas(containing 95%N₂ and 5%CO₂) for minutes,and place these cells under the condition of hypoxia for 45 minutes.The cells were harvested after reoxygenation of 90 minutes.We extracted cytosolic protein by Mitochondria/Cytosol Fractiontion Kit.The extracted Protein was stored at -70℃.The expressions of Omi/HtrA2, caspase-3/-9 were examined by western blot and the activities of caspase-3/-9 were analyzed by colorimetry.Results The expression of fluorescent protein was detected in transfected cells,but was not trailed in nontransfected cells.Compared to model groups,the expressions of Omi/HtrA2, caspase-3/-9 were significantly decreased in shRNA1 groups and shRNA2 groups(P＜0.01). Compared to normal groups,the expressions of Omi/HtrA2,caspase-3/-9 were obvious increased in model groups(P＜0.01).The expressions of Omi/HtrA2,caspase-3/-9 were no obvious change between model groups and HK groups.The expressions of Omi/HtrA2, caspase-3/-9 were no obvious change between shRNA1 groups and shRNA2 groups.The activities of caspase-3/-9 were in accordance to the expressions of caspase-3/-9 in all groups.Conclusions The Pgenesil-1/ Omi/HtrA2 shRNA1 and Pgenesil-1/ Omi/HtrA2 shRNA2 markedly inhibit on the expressions of Omi/HtrA2 of NRK-52E from hypoxia-reoxygenation injury.The caspase-9 mediated by Omi/HtrA2 was deduced,and the caspase-3 was deduced, which ultimately inhibits the apoptosis of NRK-52E.