Nrf2抑制剂4f诱导白血病细胞凋亡中miRNA-4298/PADI4信号轴的作用研究

Role of miRNA-4298/PADI4 axis in the apoptosis of leukemia cells induced by Nrf2 inhibitor 4f

目的:探讨miRNA-4298靶向PADI4调控P53在介导核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2;Nrf2)抑制剂4f诱导白血病细胞凋亡中的关键作用。方法:细胞生长密度和CCK-8计数法检测白血病细胞增殖水平;荧光定量qRT-PCR法检测细胞株中PADI4和P53基因的表达水平;流式细胞术检测白血病细胞的周期百分率和凋亡率;Western blot法检测PADI4、P53、Bcl-2、Bax、caspase-3和caspase-9的蛋白质表达水平;二代测序检测miRNA和mRNA的基因表达水平;TargetScan数据库进行靶向预测PADI4上游的miRNA和下游关联基因;荧光素酶试验检测miRNA-4298靶向结合的PADI43′UTR及其活性调控作用。细胞转染试验检测siRNA、PADI4载体、miRNA mimics和miRNA inhibitor的干扰或拯救作用。结果:Nrf2抑制剂4f具有明显抑制THP-1、K562和U937细胞增殖水平以及诱导其凋亡的作用。4f主要通过miRNA-4298的靶向海绵作用降低PADI4表达水平,继而抑制白血病细胞增殖并诱导其凋亡。进一步研究发现,上述作用与PADI4表达水平降低后的下游P53表达水平升高有关。miRNA-4298/PADI4靶向诱导P53的表达水平升高,导致下游Bcl-2蛋白表达降低和Bax蛋白表达升高。Bcl-2/Bax比值的倒置继而激活caspase-3和caspase-9蛋白,导致白血病细胞发生凋亡现象。结论:miRNA-4298/PADI4/P53信号轴是介导4f靶向诱导白血病细胞凋亡的重要机制途径,也是靶向干预的新型信号通路。

Objective To investigate the role of miRNA-4298/PADI4/p53 signal axis in mediating 4f-induced apoptosis of leukemia cells.Methods The cell growth density was observed under inverted microscope and the proliferation of leukemia cells was detected by CCK-8 counting assay.The expression of PADI4 and P53 at mRNA level was detected by qRT-PCR.Cell cycle and apoptosis were measured with flow cytometry.The expression of PADI4,P53,Bcl-2,Bax,caspase-3 and caspase-9 at protein level was detected by Western blot.Differential miRNA and mRNA expression profiles was detected by next generation sequencing.Databases such as TargetScan were used to predict the potential upstream and downstream genes of PADI4.A luciferase reporter assay was used to detect the 3′UTR of PADI4 targeted by miRNA-4298.Cell transfection assay was used to detect the effect siRNA,PADI4 vector,miRNA mimics and miRNA inhibitor in interference and rescue.Results Nuclear factor erythroid 2-related factor 2(Nrf2)inhibitor 4f could inhibit the proliferation of THP-1,K562 and U937 cells,and induce the apoptosis of these leukemia cells.It downregulated the expression of PADI4 mainly through the binding activity of miRNA-4298 to miRNA sponges,which resulted in the proliferation inhibition and apoptosis of leukemia cells.The inhibited proliferation and apoptosis of leukemia cells by 4f were associated with the increase of P53 expression after the decrease of PADI4 expression.The PADI4-dependent upregulation of P53 led to the ratio inversion of downstream Bcl-2/Bax,which activated caspase-3 or caspase-9 to induce the apoptosis of leukemia cells.Conclusions The apoptosis of leukemia cells induced by Nrf2 inhibitor 4f was mainly associated with the miRNA-4298/PADI4/p53 axis,suggesting that it might be a novel signaling pathway for targeted therapy.