亚砷酸钠及其代谢产物诱导16HBE细胞BCL2L2-PABPN1表达及机制探讨

Mechanism of BCL2L2-PABPN1 expression induced by sodium arsenite and its metabolites in 16HBE cells

目的探讨亚砷酸钠及其甲基化代谢产物诱导16HBE细胞融合基因B淋巴细胞瘤-2样蛋白2(BCL2L2)-多聚(A)结合蛋白核1(PABPN1)表达的差异及相关机制。方法(1)分别以浓度为1.5、3.0、4.5μmol/L的亚砷酸钠染毒16HBE细胞(设为低、中、高剂量砷染毒组),以浓度为4.5μmol/L一甲基胂酸(MMA)、二甲基胂酸(DMA)和亚砷酸钠染毒16HBE细胞(设为MMA组、DMA组和亚砷酸钠组),并设无毒物刺激的对照组。培养48 h后,以实时荧光定量聚合酶链式反应法(qRT-PCR)检测BCL2L2-PABPN1的表达。(2)设计两种小干扰RNA(siRNA)沉默片段转染16HBE细胞,以敲减BCL2L2-PABPN1,分别设为siRNA-1组和siRNA-2组;另设无敲减BCL2L2-PABPN1转染的对照组。培养48 h后,以qRT-PCR检测3组细胞中BCL2L2-PABPN1的表达,分别以MTS法、JC-1线粒体膜电位检测法检测细胞存活率和早期凋亡率,以Hoechest33342/碘化丙啶(PI)双重染色法观察细胞凋亡情况,以蛋白质免疫印迹法检测P53信号通路相关蛋白表达。结果(1)随着亚砷酸钠染毒剂量的增加,16HBE细胞BCL2L2-PABPN1相对表达水平增加(P<0.01);高剂量砷染毒组16HBE细胞BCL2L2-PABPN1相对表达水平分别高于对照组、低剂量砷染毒组和中剂量砷染毒组(P值均<0.05)。亚砷酸钠组16HBE细胞BCL2L2-PABPN1相对表达水平分别高于对照组、MMA组和DMA组(P值均<0.05);但对照组、MMA组和DMA组16HBE细胞BCL2L2-PABPN1相对表达水平两两比较,差异均无统计学意义(P值均>0.05)。(2)siRNA-1组、siRNA-2组16HBE细胞中BCL2L2-PABPN1相对表达水平和细胞存活率均低于对照组(P值均<0.05);但3组16HBE细胞早期凋亡率比较,差异无统计学意义(P>0.05)。Hoechest33342/PI双重染色结果显示,siRNA-1组、siRNA-2组16HBE细胞核固缩和早期凋亡细胞的数量均较对照组增多。siRNA-1组、siRNA-2组16HBE细胞中P53、Ser392位点磷酸化P53、B淋巴细胞瘤-2相关死亡启动子、P21和细胞色素C的蛋白相对表达水平均高于对照组(P值均<0.05),P53上调凋亡因子蛋白相对表达水平均低于对照组(P值均<0.05)。结论亚砷酸钠可能通过诱导融合基因BCL2L2-PABPN1的表达,阻滞16HBE细胞凋亡;其机制与激活P53信号通路有关。亚砷酸钠甲基化代谢产物MMD和DMA对BCL2L2-PABPN1的表达无影响。BCL2L2-PABPN1可能通过BCL2L2和PABPN1基因的协同作用发挥抗凋亡效应。

Objective To investigate the differential expression of the fusion gene BCL-2-like protein 2(BCL2L2)-poly(A)binding protein nuclear 1(PABPN1)induced by sodium arsenite(SA)and its methylated metabolites in 16HBE cells and the related mechanism.Methods i)The 16HBE cells exposed to SA at concentrations of 1.5,3.0 and 4.5μmol/L were set as low-,medium-and high-dose arsenic exposure groups.The 16HBE cells exposed to 4.5μmol/L monomethylarsonic acid(MMA),dimethylarsonic acid(DMA)and SA were set as MMA group,DMA group and SA group.The 16HBE cells without toxic stimulation were set as control group.After the cells were cultured for 48 hours,the expression of BCL2L2-PABPN1 was detected by quantitative real-time polymerase chain reaction(qRT-PCR).ii)Two small interfering RNA(siRNA)silencing fragments were designed and transfected into 16HBE cells to knockdown BCL2L2-PABPN1,which were set as siRNA-1 group and siRNA-2 group.Non-transfected control group without knockdown of BCL2L2-PABPN1 transfection was set up.After culturing for 48 hours,the expression level of BCL2L2-PABPN1 in the three groups of cells was detected by qRT-PCR.The cell survival rate and early apoptosis rate were detected by MTS method and JC-1 mitochondrial membrane potential detection method,respectively.The apoptosis was detected by Hoechest33342/propidium iodide(PI)double staining,and the expression level of P53 signaling pathway-related proteins was detected by Western blotting.Results i)The relative expression of BCL2L2-PABPN1 in 16HBE cells increased with the increasing SA doses(P<0.01).The relative expression of BCL2L2-PABPN1 in high-dose arsenic exposure was higher than that in control group,low-dose and medium-dose arsenic exposure groups(all P<0.05).The relative expression of BCL2L2-PABPN1 in SA group was higher than those in control group,MMA group and DMA group(all P<0.05).The relative expression of BCL2L2-PABPN1 showed no significant difference between control group,MMA group and DMA group(all P>0.05).ii)The relative expression levels of BCL2L2-PABPN1 and cell survival rate in siRNA-1 group and siRNA-2 group were lower than those in non-transfected control group(all P<0.05).However,there was no significant difference in the early apoptosis rate among the three groups(P>0.05).The results of Hoechest33342/PI double staining showed that the number of nuclear shrinkage and early apoptotic cells in siRNA-1 group and siRNA-2 group was higher than that in non-transfected control group.The relative protein expression levels of P53,phosphop-53,BCL-2-associated death promoter,P21 and cytochrome C in siRNA-1 group and siRNA-2 group were higher(all P<0.05),and the relative protein expression levels of P53-up-regulated modulator of apoptosis were lower(all P<0.05),when compared with the non-transfected control group.Conclusion SA may block the apoptosis of 16HBE cells by inducing the expression of fusion gene BCL2L2-PABPN1.The mechanism may be related to the activation of P53 signaling pathway.The SA methylated metabolites MMD and DMA had no effect on the expression of BCL2L2-PABPN1.BCL2L2-PABPN1 may affect anti-apoptosis through affecting the synergistic effect of BCL2L2 and PABPN1 genes.