砷及其代谢物对BCL-2基因转录本BCL-2α和BCL-2β表达的影响

Effects of arsenic and its metabolites on expressions of BCL-2α and BCL-2β transcripts

[背景]砷是一种毒物,会影响细胞抗凋亡基因BCL-2及其蛋白质的表达,但砷对BCL-2不同亚型转录本BCL-2α和BCL-2β的影响尚无相关报道。[目的]研究砷及其代谢物一甲基胂酸(MMA)和二甲基胂酸(DMA)对人正常支气管上皮样细胞(16HBE)和人肺腺癌细胞(A549)中BCL-2基因转录本BCL-2α、BCL-2β及其总和BCL-2T的影响。[方法]将16HBE细胞和A549细胞经体外培养后随机分成3大组:等浓度的不同砷化合物(MMA、DMA和亚砷酸钠)单独染毒组(16HBE细胞的处理浓度为4.5μmol·L^(-1),A549细胞为60μmol·L^(-1)),不同浓度的亚砷酸钠单独染毒组(16HBE细胞的处理浓度为1.5、3.0、4.5μmol·L^(-1),A549细胞为20、40、60μmol·L^(-1))以及联合染毒组(即MMA+亚砷酸钠、DMA+亚砷酸钠;16HBE细胞的染毒浓度分别均为1.5μmol·L^(-1)和均为4.5μmol·L^(-1),A549细胞的染毒浓度分别均为20μmol·L^(-1)及60μmol·L^(-1)),同时在各染毒组中均设立空白对照组。持续染毒48 h后采用实时荧光定量PCR检测两种细胞中BCL-2α、BCL-2β和BCL-2T的相对表达情况。[结果]不同砷化合物单独染毒:16HBE细胞在4.5μmol·L^(-1)MMA处理下,BCL-2α和BCL-2T的表达水平均低于对照组(q=3.27、2.93,均P<0.05),在4.5μmol·L^(-1)亚砷酸钠处理下BCL-2α、BCL-2β和BCL-2T的表达水平均低于各自对照组(q=11.06、3.65、10.70,均P<0.05)。A549细胞在60μmol·L^(-1)DMA处理下BCL-2T的表达水平低于对照组(q=3.12,P<0.05),在60μmol·L^(-1)亚砷酸钠处理下BCL-2α、BCL-2β和BCL-2T的表达水平均低于各自对照组(q=7.59、7.27、8.06,均P<0.05)。亚砷酸钠单独染毒:16HBE细胞在1.5μmol·L^(-1)亚砷酸钠处理下,BCL-2α的表达水平低于对照组,BCL-2β的表达水平高于对照组(q=6.06、11.92,均P<0.05);在3.0μmol·L^(-1)亚砷酸钠处理下,BCL-2α的表达水平低于对照组(q=12.72,P<0.05);在4.5μmol·L^(-1)亚砷酸钠处理下,BCL-2α、BCL-2β和BCL-2T的表达水平均低于各自对照组(q=15.72、6.79、6.62,均P<0.05)。BCL-2α的表达水平随亚砷酸钠浓度升高逐渐下降(F_(α趋势)=144.80,P<0.001),BCL-2β和BCL-2T则在1.5~4.5μmol·L^(-1)范围内呈剂量依赖性降低(F_(β趋势)=135.40,F_(T趋势)=38.24,均P<0.001)。在各浓度亚砷酸钠处理下A549细胞BCL-2α、BCL-2β和BCL-2T的表达水平均低于各自对照组(均P<0.05);且进一步趋势检验结果显示,BCL-2α、BCL-2β和BCL-2T的表达水平均随亚砷酸钠浓度增加逐渐下降(F_(α趋势)=31.97,F_(β趋势)=549.50,F_(T趋势)=252.40,均P<0.001)。联合染毒:在均为60μmol·L^(-1)的MMA+亚砷酸钠处理下,A549细胞BCL-2α、BCL-2β和BCL-2T的表达水平均高于对照组(q=6.37、14.91、5.33,均P<0.05);在均为60μmol·L^(-1)的DMA+亚砷酸钠处理下,A549细胞BCL-2α、BCL-2β和BCL-2T的表达水平也均高于各自对照组(q=8.60、17.29、6.91,均P<0.05)[结论]高浓度(16HBE:4.5μmol·L^(-1);A549:60μmol·L^(-1))的砷代谢产物单独染毒对16HBE及A549细胞的BCL-2转录本多无影响。低浓度(1.5μmol·L^(-1))的亚砷酸钠单独染毒可降低16HBE细胞BCL-2α的表达水平,升高其BCL-2β的表达水平;各浓度亚砷酸钠单独染毒可降低A549细胞中所有转录本的表达水平。高浓度(60μmol·L^(-1))的MMA与亚砷酸钠联合染毒以及高浓度(60μmol·L^(-1))的DMA与亚砷酸钠联合染毒均会升高A549细胞中BCL-2α、BCL-2β和BCL-2T的表达水平,与单独染毒呈现的效应不同。

[Background]Arsenic is a toxicant that can affect the expressions of the cellular anti-apoptotic gene BCL-2 and its protein,but the effects of arsenic on BCL-2αand BCL-2βtranscripts have not been reported.[Objective]To investigate the potential effects of arsenic and its metabolites,methylarsonic acid(MMA)and dimethylarsonic acid(DMA),on BCL-2α,BCL-2β,and BCL-2 T(total ofαandβtranscripts)in human bronchial epithelial cells(16 HBE)and human lung adenocarcinoma cells(A549).[Methods]16 HBE cells and A549 cells were randomly divided into three categories of exposure after in vitro culture:single-selected arsenic compound exposure groups with isoconcentration(16 HBE cells were treated with 4.5μmol·L^(-1)of MMA,DMA,and sodium arsenite,respectively,while A549 cells were treated with 60μmol·L^(-1)of MMA,DMA,and sodium arsenite,respectively),sodium arsenite exposure groups with different concentrations(16 HBE cells were treated with 1.5,3.0,and 4.5μmol·L^(-1)of sodium arsenite respectively,while A549 cells were treated with 20,40,and 60μmol·L^(-1)of sodium arsenite respectively),and combined exposure groups(i.e.MMA+sodium arsenite,and DMA+sodium arsenite;the exposure concentrations of 16 HBE cells were both 1.5μmol·L^(-1)and both4.5μmol·L^(-1)respectively,and those of A549 cells were both 20μmol·L^(-1)and both 60μmol·L^(-1)respectively).Meanwhile,a blank control group was also set up in each exposure category.After 48 h of continuous exposure,the relative expressions of BCL-2α,BCL-2β,and BCL-2 T in both cells were detected by real-time PCR.[Results]Regarding the single-selected arsenic compound exposure,in 16 HBE cells,the expression levels of BCL-2αand BCL-2 T under4.5μmol·L^(-1)MMA treatment were lower than those in their control groups(q=3.27,2.93,both P<0.05),and the expression levels of BCL-2α,BCL-2β,and BCL-2 T under 4.5μmol·L^(-1)sodium arsenite were lower than those in their respective control groups(q=11.06,3.65,10.70,all P<0.05).In A549 cells,the expression level of BCL-2 T treated with 60μmol·L^(-1)DMA was lower than that in the control group(q=3.12,P<0.05),and the expression levels of BCL-2α,BCL-2β,and BCL-2 T treated with 60μmol·L^(-1)sodium arsenite were lower than those in their respective control groups(q=7.59,7.27,8.06,all P<0.05).Regarding the sodium arsenite exposure:16 HBE cells treated with 1.5μmol·L^(-1)sodium arsenite had a lower expression level of BCL-2αand a higher expression level of BCL-2βthan those in their respective control groups(q=6.06,11.92,both P<0.05);the expression level of BCL-2αunder 3.0μmol·L^(-1)sodium arsenite was lower than that in the control group(q=12.72,P<0.05);and under 4.5μmol·L^(-1)sodium arsenite treatment,the expression levels of BCL-2α,BCL-2β,and BCL-2 T were lower than those in their respective control groups(q=15.72,6.79,6.62,all P<0.05).The expression levels of BCL-2αgradually decreased with increasing concentrations of sodium arsenite(F_(αtrend)=144.80,P<0.001),while BCL-2βand BCL-2 T decreased in a dose-dependent manner in the range of 1.5-4.5μmol·L^(-1)(F_(βtrend)=135.40,F_(T trend)=38.24,both P<0.001).In A549 cells,the expression levels of BCL-2α,BCL-2β,and BCL-2 T under each concentration of sodium arsenite treatments were lower than those in their respective control groups(all P<0.05);the results of further trend tests showed that their expression levels gradually decreased with increasing concentrations of sodium arsenite(F_(αtrend)=31.97,F_(βtrend)=549.50,F_(T trend)=252.40,all P<0.001).Regarding the combined exposure,under MMA+sodium arsenite treatment at both 60μmol·L^(-1),the expression levels of BCL-2α,BCL-2β,and BCL-2 T in A549 cells were higher than those in their respective control groups(q=6.37,14.91,5.33,all P<0.05);under DMA+sodium arsenite treatment at both 60μmol·L^(-1),their expression levels in A549 cells were also higher than those in their respective control group(q=8.60,17.29,6.91,all P<0.05).[Conclusion]Exposure to a high concentration(16 HBE:4.5μmol·L^(-1),A549:60μmol·L^(-1))of a single arsenic metabolite has no effect on BCL-2 transcripts in 16 HBE cells and A549 cells.Exposure to a low concentration(1.5μmol·L^(-1))of sodium arsenite alone would decrease the expression level of BCL-2αand increase the expression level of BCL-2βin 16 HBE cells,and exposure to all designed concentrations of sodium arsenite alone would decrease the expressions of all transcripts in A549 cells.The combined exposure to high concentrations(both 60μmol·L^(-1))of MMA plus sodium arsenite or high concentrations(both 60μmol·L^(-1))of DMA plus sodium arsenite would increase the expressions of BCL-2α,BCL-2β,and BCL-2 T in A549 cells,which are different from the effects presented by single exposure.