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  • Title: [Mechanism of nuclear translocation of hypoxia-inducible factor-1α in influenza A (H1N1) virus infected-alveolar epithelial cells].
    Author: Meng X, Guo X, Zhu Y, Xie H, Wang R.
    Journal: Zhonghua Wei Zhong Bing Ji Jiu Yi Xue; 2020 Jan; 32(1):8-13. PubMed ID: 32148224.
    Abstract:
    OBJECTIVE: To investigate the molecule mechanism of nuclear translocation of hypoxia-inducible factor-1α (HIF-1α) in influenza A (H1N1) virus infected-alveolar epithelial cells. METHODS: Human lung adenocarcinoma epithelial cells (A549 cells) were cultured in vitro, and cells in logarithmic growth phase were selected for experiments. (1) Experiment 1: the A549 cell model with H1N1 virus infection was established by using H1N1 virus infected cells with multiplicity of infection (MOI) 1.0 for 24 hours (H1N1 virus infection group), and the blank control group was set up. Importin 4 and Importin 7 protein expressions were detected by Western Blot to investigate whether HIF-1α nuclear translocation depended on Importin 4 or Importin 7. (2) Experiment 2: the A549 cells were infected with H1N1 virus under different MOI (0, 0.1, 0.5, 1.0, 2.0, 4.0) for 24 hours. Then the A549 cells were infected with H1N1 virus (MOI 1.0) for different time (0, 3, 6, 12, 18, 24, 36 hours). The septin 9 isoform 1 (SEPT9_i1) mRNA expression was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) to investigate the effect of different MOI and infection time on the expression of SEPT9_i1. (3) Experiment 3: a cell model with SEPT9_i1 silencing was established by transfection of small interfering RNA (siRNA) for 24 hours (siRNA-SEPT9_i1 group), and the blank control group and blank vector control group (siControl group) were set up. Then the cells in the three groups were infected with H1N1 virus (MOI 1.0) for 24 hours after 24-hour transfection, and the SEPT9_i1 mRNA expression was detected by real-time fluorescence quantitative RT-PCR to investigate the interference efficiency of siRNA-SEPT9_i1. (4) Experiment 4: the cells were divided into siControl group and siRNA-SEPT9_i1 group. The transfection methods of two groups was as the same as experiment 3,and then the cells were infected with H1N1 virus (MOI 1.0) after 24-hour transfection. The distribution of HIF-1α was detected by immunofluorescence at 24 hours after infection. The M gene expression of virus was detected by real-time fluorescence quantitative RT-PCR at 6, 12, 24, 36, 48 hours after infection. The effects of SEPT9_i1 on HIF-1α translocation and virus replication were explored. (5) Experiment 5: the cells were divided into blank control group (complete medium), SP600125 group [100 μmol/L c-Jun N-terminal kinase (JNK) signaling pathway inhibitor SP600125 for 2 hours], H1N1 virus infection group (H1N1 virus of MOI 1.0 for 24 hours), H1N1 virus+SP600125 group (pretreated with 100 μmol/L SP600125 for 2 hours before 24-hour H1N1 virus infection). Real-time fluorescence quantitative RT-PCR was used to detect the expressions of SEPT_i1 mRNA and viral M gene to investigate the effect of JNK signaling pathway on SEPT9_i1 expression and virus replication. RESULTS: (1) Experiment 1: compared with the blank control group, the protein expressions of Importin 4 and Importin 7 in the H1N1 virus infection group had no significant changes [Importin 4 protein (Importin 4/GAPDH): 1.08±0.03 vs. 1.05±0.03, Importin 7 protein (Importin 7/GAPDH): 0.87±0.11 vs. 0.78±0.03, both P > 0.05]. These indicated that the HIF-1α nuclear translocation in A549 cells might not be independent of Importin 4 and Importin 7 during H1N1 virus infection. (2) Experiment 2: the SEPT9_i1 mRNA expression in A549 cells was increased with the increase in MOI and infection time of H1N1 virus, and peaked at MOI 2.0 or 18 hours after infection, and the differences were statistically significant as compared with MOI 0 or 0 hour after infection (2-ΔΔCT: 1.39±0.05 vs. 1.00±0.00 at MOI 2.0, 1.47±0.04 vs. 1.00±0.00 at 18 hours, both P < 0.01). This indicated that the SEPT9_i1 expression in A549 cells was related to the MOI and the infection time during H1N1 virus infection. (3) Experiment 3: compared with the blank control group, the SEPT9_i1 mRNA expression in A549 cells was significantly decreased in the siRNA-SEPT9_i1 group (2-ΔΔCT: 0.38±0.11 vs. 1.00±0.00, P < 0.01), and there was no significant difference between the siControl group and blank control group (2-ΔΔCT: 1.03±0.16 vs. 1.00±0.00, P > 0.05). This indicated that SEPT9_i1 silence could inhibit the expression of SEPT9_i1 mRNA in H1N1 virus-infected A549 cells. (4) Experiment 4: HIF-1α nuclear translocation in the H1N1 virus-infected A549 cells in the siRNA-SEPT9_i1 group was significantly reduced as compared with the siControl group. The virus M gene expression after H1N1 virus infection in the siControl group was gradually increased, and peaked at 48 hours. The expression of virus M gene in A549 cells in the siRNA-SEPT9_i1 group was significantly down-regulated, and showed a statistically significant difference at 48 hours as compared with the siControl group (2-ΔΔCT: 3.47±0.66 vs. 8.17±0.38, P < 0.05). This indicated that HIF-1α nuclear translocation and virus replication in H1N1 virus-infected A549 cells were inhibited after silencing SEPT9_i1. (5) Experiment 5: the expressions of SEPT9_i1 mRNA and virus M gene in A549 cells in the H1N1 virus infection group were significantly higher than those in the blank control group. However, the expressions of SEPT9_i1 mRNA and viral M gene in A549 cells in the H1N1 virus+SP600125 group were significantly lower than those in the H1N1 virus infection group (2-ΔΔCT: SEPT9_i1 mRNA was 0.12±0.10 vs. 1.53±0.14, viral M gene was 2.13±0.10 vs. 4.66±0.14, both P < 0.05). There was no significant difference in above indicators between the SP600125 group and the blank control group. This indicated that the JNK signaling pathway could regulate the expression of SEPT9_i1 in A549 cells during H1N1 virus infection, and the JNK signaling pathway inhibition could down-regulate the expression of SEPT9_i1 and inhibit virus replication. CONCLUSIONS: The H1N1 virus regulates the expression of SEPT9_i1 by activating the JNK signaling pathway, thus increase HIF-1α transport efficiency and H1N1 replication.
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