HSP70 silencing aggravates apoptosis induced by hypoxia/reoxygenation in vitro .
EXPERIMENTAL AND THERAPEUTIC MEDICINE(2019)
Abstract
Lung ischemia-reperfusion can cause acute lung injury, which is closely associated with apoptosis. Heat shock protein 70 (HSP70) is an anti-apoptotic protein that promotes cell survival under a variety of different stress conditions. However, the role and mechanism of HSP70 in lung ischemia-reperfusion injury is yet to be fully elucidated. In the present study, an in vitro hypoxia/reoxygenation model of A549 cells was established to simulate lung ischemia-reperfusion and HSP70 was silenced by transfecting A549 cells with an shRNA sequence targeting HSP70. Western blotting, reverse transcription-quantitative polymerase chain reaction, Cell Counting kit-8 and flow cytometry were used to detect protein levels, RNA expression, cell activity and apoptosis. The results revealed that silencing HSP70 reduced cell viability, aggravated apoptosis, increased lactate dehydrogenase levels and induced a G2/M blockade in a hypoxia-reoxygenation A549 cell model. Furthermore, silencing HSP70 decreased the phosphorylation levels of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK); however, the total AKT and ERK levels did not change significantly. Pretreating A549 cells with the AKT pathway inhibitor, LY294002 and the ERK pathway inhibitor, U0216 led to a decrease in HSP70 expression. These results indicate that silencing HSP70 may aggravate apoptosis in hypoxia-reoxygenation cell models, potentially via the mitogen-activated protein kinase/ERK and phosphoinositide 3-kinase/AKT signaling pathways.
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Key words
heat shock protein 70,apoptosis,hypoxia,reoxygenation,phosphoinositide 3-kinase,protein kinase B,mitogen-activated protein kinase,extracellular signal-regulated kinase
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