miR-155/BACH1信号通路在三氧化二砷诱导肺腺癌细胞死亡中的机制研究

miR-155/BACH1 Signaling Pathway in Human Lung Adenocarcinoma Cell Death Induced by Arsenic Trioxide

目的探讨微小RNA 155(miR-155)、BTB和CNC同源蛋白1(BACH1)、醌氧化还原酶1(NQO1)和血红素氧合酶-1(HO-1)在三氧化二砷(ATO)诱导细胞死亡过程中的变化规律,以及miR-155和BACH1可能的调控关系,为增敏ATO治疗新靶点的发现奠定实验基础。方法以不同浓度ATO处理肺腺癌A549细胞后,采用MTT实验检测细胞的存活率或死亡率,试剂盒检测细胞的总抗氧化能力,Westom blot检测BACH1、NQO1和HO-1蛋白的表达,实时荧光定量PCR(qRT-PCR)检测miR-155的表达水平。miR-155类似物(mimic)及其阴性对照转染对数期A549细胞,并设未转染组为对照,qRT-PCR检测miR-155表达水平后,以20μmol/L ATO处理24h,再进行MTT及Western blot检测。结果 10~100μmol/L的ATO可降低细胞的存活率;与空白对照组相比,10、20μmol/L的ATO可减弱细胞的总抗氧化能力,激活BACH1蛋白的表达,抑制miR-155以及NQO1和HO-1蛋白的表达;在转染miR-155mimic后,20μmol/L ATO处理后的A549细胞死亡率低于未转染组和转染阴性对照组,且ATO对BACH1蛋白的激活作用减弱,NQO1和HO-1蛋白表达则高于后两组(P<0.05)。结论ATO可通过抑制miR-155的表达,激活BACH1蛋白,抑制NQO1和HO-1蛋白的表达,从而削弱细胞的总抗氧化能力,最终诱导细胞死亡,提示miR-155和BACH1可作为ATO治疗肺癌过程中的增敏靶点。

Objective To explore the changes of micro RNA 155 （miR-155）, BTB and CNC homologous protein 1 （BACH1）, quinone oxidoreductase 1 （NQO1） and heme-oxygenase-1 （HO-1） in the process of arsenic trioxide-induced cell death, and to clarify the relationship between miR-155 and BACH1, providing experimental basis for the sensitivity of arsenic trioxide （ATO） treatment. Methods Human lung adenocarcinoma cell line A549 cells were treated with different concentrations of ATO. MTT assay and total antioxidant capacity detection kit were used to determine cell viability and total antioxidant capacity, respectively. BACH1, NQO1 and HO-1 protein expression were probed by Western blot and real-time fluorescence quantitative （qRT-PCR） was utilized to test the miR-155 level. A549 cells were transfected with miR-155 mimic and its negative control, then the expression level of miR-155 was detected by qRT-PCR, and these cells were treated with 20 μmol/L for 24 h followed by MTT and Western blot detection. Results 10 μmol/L ATO significantly reduced the cell viability in A549 cells. 10 μmol/L and 20 μmol/L ATO treatment activated BACH1 expression and inhibited miR-155, NQO1 and HO-1 expression, leading to decreased total antioxidant capacity. Importantly, the cell death induced by 20 μmol/L ATO was significantly decreased in miR-155 mimic transfection cells in comparison with non-transfected cells and miR-155 mimic negative control transfected cells. Moreover, high expression of miR-155 reduced BACH1 activation and increased NQO1 and HO-1 expression in cells treated with 20 μmol/L ATO （ P〈0.05）. Conclusion Restraining total antioxidant capacity contributes to ATO induced cell death, the underlying mechanisms may be that ATO can activate BACH1 expression through inhibition of the miR-155 level, leading to subsequent inhibition of NQO1 and HO-1 expression. Taken together, these data suggest that miR-155 and BACH1 could be used as sensitivity targets for ATO treatment in lung cancer.