下调GPX1通过MAPK/NF-κB通路抑制急性髓系白血病细胞增殖和迁移

李一丹; 杨艳敏; 董照

下调GPX1通过MAPK/NF-κB通路抑制急性髓系白血病细胞增殖和迁移

Down regulation of GPX1 inhibits proliferation and migration of acute myeloid leukemia cells through MAPK/NF-κB pathway

摘要

摘要:
目的： 探究谷胱甘肽过氧化物酶1（GPX1）对急性髓系白血病（AML）细胞株增殖、迁移及丝裂素活化蛋白激酶（MAPK）/核因子-κB（NF-κB）信号通路的影响。
方法： 体外培养不同细胞系（HS-5、HL-60、KG-1、kasumi-1和THP-1），将KG-1细胞分为A组和B组，A组分为沉默GPX1阴性对照（si-NC）组、沉默GPX1-1（si-GPX1-1）组、沉默GPX1-2（si-GPX1-2）组和沉默GPX1-3（si-GPX1-3）组；B组分为空白对照（Control）组、沉默GPX1阴性对照（si-NC）组、沉默GPX1-1（si-GPX1-1）组、沉默GPX1-1和MAPK/NF-κB信号通路激动剂（si-GPX1-1 + PMA）组。实时荧光定量逆转录聚合酶链反应和Western blotting检测不同细胞系和A组KG-1细胞中GPX1相对表达水平；CCK-8和EdU法检测B组KG-1细胞增殖情况；划痕实验和Transwell实验检测B组KG-1细胞迁移情况；Western blotting检测B组KG-1细胞中增殖、迁移及MAPK/NF-κB信号通路相关蛋白（PCNA、CyclinD1、MMP-2、MMP-9、p-ERK、ERK、p-JNK、JNK、p-p38、p38、p-IκB、IκB、p-p65和p65）水平。
结果： 与人骨髓基质细胞HS-5相比，GPX1 mRNA和蛋白水平在AML细胞（HL-60、KG-1、kasumi-1和THP-1）中上调（P ＜ 0.05），且KG-1细胞中表达水平最高。与si-NC组相比，A组其余3组KG-1细胞中GPX1 mRNA和蛋白表达水平均降低（P ＜ 0.05），且si-GPX1-1组沉默效率高于其他两组。与si-NC组相比，si-GPX1-1组KG-1细胞增殖和迁移能力均降低（P ＜ 0.05）；细胞中PCNA、CyclinD1、MMP-2、MMP-9蛋白水平降低（P ＜ 0.05）；p-ERK/ERK、p-JNK/JNK、p-p38/p38、p-IκB/IκB和p-p65/p65比值下调（P ＜ 0.05）。与si-GPX1-1组相比，si-GPX1-1 + PMA组KG-1细胞增殖和迁移能力均增高（P ＜ 0.05）；细胞中PCNA、CyclinD1、MMP-2、MMP-9蛋白水平增高（P ＜ 0.05）；p-ERK/ERK、p-JNK/JNK、p-p38/p38、p-IκB/IκB和p-p65/p65比值上调（P ＜ 0.05）。
结论： 下调GPX1可能通过抑制MAPK/NF-κB信号通路抑制AML细胞增殖和迁移。

Abstract:
Objective To explore the influences of glutathione peroxidase 1 (GPX1) on the proliferation, migration and mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) signal pathway of acute myelogenous leukemia (AML) cell lines.
Methods The cell lines (HS-5, HL-60, KG-1, kasumi-1 and THP-1) were cultured in vitro. The KG-1 cells were divided into group A and group B. Among them, the group A included: silent GPX1 negative control (si-NC) group, silent GPX1-1 (si-GPX1-1) group, silent GPX1-2 (si-GPX1-2) group, and silent GPX1-3 (si-GPX1-3) group; the group B included: blank control (Control) group, silent GPX1 negative control (si-NC) group, silent GPX1-1 (si-GPX1-1) group, silent GPX1-1 and MAPK/NF-κB signaling pathway agonist (si-GPX1-1 + PMA) group. Real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting were used to detect the relative expression level of GPX1 in different cell lines and group A cells. CCK-8 and EdU methods were used to detect the proliferation of KG-1 cells in group B. Scratch test and transwell test were used to detect the migration of KG-1 cells in group B. Western blotting was used to detect the levels of proliferation, migration and MAPK/NF-κB signaling pathway related proteins (PCNA, CyclinD1, MMP-2, MMP-9, p-ERK, ERK, p-JNK, JNK, p- p38, p38, p-IκB, IκB, p-p65 and p65).
Results Compared with human bone marrow stromal cells HS-5, the GPX1 mRNA and protein levels were up-regulated in AML cells (HL-60, KG-1, kasumi-1 and THP-1) (P ＜ 0.05), and highest in KG-1 cells. Compared with the si-NC group, the expression of GPX1 mRNA and protein in the KG-1 cells of the remaining three groups of group A was reduced (P ＜ 0.05), and the silencing efficiency of the si-GPX1-1 group was higher than that of the other two groups. Compared with the si-NC group, the proliferation and migration abilities of KG-1 cells in the si-GPX1-1 group were reduced (P ＜ 0.05); the protein levels of PCNA, CyclinD1, MMP-2, and MMP-9 in cells were decreased (P ＜ 0.05); the ratios of p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-IκB/IκB and p-p65/p65 were down-regulated (P ＜ 0.05). Compared with the si-GPX1-1 group, the proliferation and migration abilities of KG-1 cells in the si-GPX1-1 + PMA group were increased (P ＜ 0.05); the protein levels of PCNA, CyclinD1, MMP-2, and MMP-9 in cells were increased (P ＜ 0.05); the ratios of p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-IκB/IκB and p-p65/p65 were up-regulated (P ＜ 0.05).
Conclusion Down-regulation of GPX1 may inhibit the proliferation and migration of AML cells through inhibiting the MAPK/NF-κB signaling pathway.

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