HSP70 silencing aggravates apoptosis induced by hypoxia/reoxygenation in vitro

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.

Keywords: apoptosis; extracellular signal-regulated kinase; heat shock protein 70; hypoxia; mitogen-activated protein kinase; phosphoinositide 3-kinase; protein kinase B; reoxygenation.

Figure 1.

Lentivirus transfection efficiency detected via fluorescence microscopy. A549 cells were transfected with the recombinant lentivirus containing the shRNA sequence of HSP70 (magnification, ×100). Green fluorescence was observed in successfully transfected A549 cells. A549 Cells after 48 h of infection under (A) white and (B) fluorescent light. The infection efficiency was ~80%. shRNA, short hairpin RNA; HSP70, heat shock protein 70.

Figure 2.

Expression of HSP70 mRNA and protein in A549 cells. The (A) mRNA and (B) protein expression of HSP70 was determined via reverse transcription-quantitative polymerase chain reaction and western blotting. (C) The result of western blotting was quantified three times. *P<0.05 vs. the non-infection group; ^#P<0.05 vs. the lentivirus control group. HSP70, heat shock protein 70.

Figure 3.

A549 cell viability as determined via a cell CCK-8 assay following different durations of hypoxia and reoxygenation for 24 h. The viability of the lentivirus infection group was significantly lower than that of the lentivirus control and non-infection groups at all time points. Data are presented as the mean ± standard deviation. *P<0.05 vs. the non-infection group; ^#P<0.05 vs. the lentivirus control group. CCK, cell counting kit.

Figure 4.

A549 cells apoptosis following hypoxia/reoxygenation. Following A549 cell exposure to hypoxia for 6 h and then reoxygenation for 24 h, the apoptosis rate of cells was measured via flow cytometry with Annexin V-allophycocyanin/propidium iodide. Representative (A) flow cytometry results and (B) apoptosis ratio. Data are presented as the mean ± standard deviation. *P<0.05 vs. the non-infection group; ^#P<0.05 vs. the lentivirus control group.

Figure 5.

Effect of HSP70 silencing on the protein expression of caspase-3, Bcl-2 and Bax. (A) Western blotting was performed to assess (B) caspase-3 and (C) Bcl-2 and Bax protein levels. Data are presented as the mean ± standard deviation *P<0.05 vs. the non-infection group; ^#P<0.05 vs. the lentivirus control group. HSP70, heat shock protein 70; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X.

Figure 6.

HSP70 silencing arrests G2/M phase. Cell cycle distribution was detected using a flow cytometer following A549 cell exposure to hypoxia for 6 h and reoxygenation for 24 h. (A) Flow cytometry results and (B) the ratio of G2/M phase cells are presented. *P<0.05 vs. the non-infection group; ^#P<0.05 vs. lentivirus control group. HSP70, heat shock protein 70; 2N, G1 phase; 4N, G2/M phase.

Figure 7.

HSP70 silencing promotes the release of LDH. Following A549 cell exposure to hypoxia/reoxygenation for 6 and 24 h respectively, cell culture medium was collected for LDH determination. lentivirus control (^#P<0.05 vs. the lentivirus control group; *P<0.05 vs. the non-infection group. HSP70, heat shock protein 70; LDH, lactate dehydrogenase.

Figure 8.

Effect of HSP70 silencing on the mitogen-activated protein kinase/ERK signaling pathways. A549 cells were exposed to hypoxia for 6 h with or without 10 µM U0126. (A) Western blotting was performed to determine the protein expression of HSP70, p-ERK and total ERK. GADPH was used as the loading control. Following western blotting, the ratios of (B) HSP70/GAPDH and (C) p-ERKERK were calculated. *P<0.05 vs. the non-infection group; ^#P<0.05 vs. the lentivirus control group. HSP70, heat shock protein 70; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; p-, phosphorylated.

Figure 9.

Effect of HSP70 silencing on the PI3K/AKT signaling pathway. A549 cells were exposed to hypoxia for 6 h with or without 50 µM LY294002. (A) Western blotting was performed to determine the protein expressions of HSP70, p-AKT and total AKT. GADPH was used as the loading control. Following western blotting, the ratios of (B) HSP70/GAPDH and (C) p-AKTtotal-AKT were calculated. ^#P<0.05 vs. the lentivirus control group; *P<0.05 vs. the non-infection group. HSP70, heat shock protein 70; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; p-, phosphorylated.

Figure 10.

Inhibition of the AKT and ERK signaling pathway results in the decreased expression of HSP70 mRNA. Following A549 cell exposure to hypoxia for 6 h with or without 10 µM U0126 or 50 µM LY294002, the mRNA levels of HSP70 were detected via reverse transcription-quantitative polymerase chain reaction. ^#P<0.05 vs. the lentivirus control group; *P<0.05 vs. the non-infection group. AKT, protein kinase B; ERK, extracellular signal-regulated kinase; HSP70, heat shock protein 70.