敲减AKR1A1基因对H_2O_2及4-羟基壬烯醛诱导的1321N1脑星形细胞瘤细胞损伤的影响

Effect of AKR1A1 knock-down on H_2O_2 and 4-hydroxynonenal-induced cytotoxicity in human 1321N1 astrocytoma cells

目的初步探讨星形胶质细胞瘤细胞中的醛酮还原酶1A1(AKR1A1)在抗氧化应激和有毒醛代谢中的作用。方法通过LipofectamineTM RNAiMax以siRNA转染1321N1细胞,用Western blot法和qRT-PCR检测1321N1细胞中AKR1A1基因抑制水平。siRNA转染后的细胞经H2O2和4-羟基壬烯醛处理后使用MTT法检测细胞成活率;采用2',7'-二氯二氢荧光黄双乙酸酯(DCFH-DA)标记法检测敲减AKR1A1基因对H2O2诱导的1321N1细胞内活性氧(ROS)水平的影响。结果 Western blot法和qRT-PCR结果显示1321N1细胞经特异性siRNA转染后,AKR1A1基因的表达受到明显抑制(70%)。MTT法检测结果显示,siRNA-AKR1A1转染后的1321N1细胞在H2O2或4-羟基壬烯醛压力下的细胞成活率显著降低,而且敲减AKR1A1的1321细胞内H2O2诱导的ROS水平显著高于对照细胞。结论使用的特异性siRNA能有效抑制AKR1A1基因在1321N1星形细胞瘤细胞中表达,AKR1A1参与1321N1脑星形细胞瘤细胞中的4-羟基壬烯醛代谢,并且很可能参与调节脑细胞的抗氧化应激机制。

Objective To investigate the role of human aldo-keto reductase ]A1 （AKRIAI） in the resistance to oxidative stress and the metabolism of toxic aldehyde in astrocytoma cells. Methods The siRNA was transfected into 1321N1 astrocy- toma cells using LipofectamineTM RNAiMax. Western blotting and qRT-PCR were applied to evaluate the knock-down efficien- cy of AKR1AI. MFT assay was used to examine the cell viability after H202 and 4-hydroxynonenal treatment in AKFI1AI knock-down cells. In addition, the effect of knocking down AKR1AI on cellular reactive oxygen species （ROS） level in the presence of H202 was measured using 2＇, ？＇-dichlorofluorescein （DCFH-DA）. Results Western blotting and qRT-PCR showed that the AKRlAl-specific siRNA inhibited AKRIA] gene expression by about 70% in 1321N1 cells. Cells with knock- down of AKRIAI were more sensitive to H202 and 4-hydroxynonenal-induced cytotoxicity. Furthermore, cellular ROS level in the cells with knock-down of AKR1AI was much higher than that in the control cells in the presence of H202. Conclusion The specific siRNA could efficiently inhibit AKR1AI expression in 1321N1 cells. AKRIAI could be involved in the metabolism of 4-hydroxynonenal and play a role in the resistance to oxidative stress.