异柠檬酸脱氢酶1基因突变对替莫唑胺干预下脑胶质瘤U87细胞凋亡的影响

Mechanisms of mIDH1 enhancing the therapeutic sensitivity of temozolomide by interfering with U87 cell apoptosis

目的研究异柠檬酸脱氢酶1(isocitrate dehydrogenase 1,IDH1)基因突变对替莫唑胺干预下脑胶质瘤U87细胞凋亡的影响,以期深入了解其生物学特性及其化疗敏感性。方法通过DNA重组技术构建携带野生IDH1基因(wIDH1)或突变的IDH1基因(mIDH1)的真核表达载体;通过CCK-8检测三种稳定细胞系的增殖能力和细胞活力;利用流式细胞仪技术检测稳转细胞系细胞凋亡率及替莫唑胺干预后细胞凋亡率的变化;构建裸鼠移植瘤模型,替莫唑胺干预,ELISA技术和蛋白质免疫印迹检测细胞相关凋亡蛋白[半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、B淋巴细胞瘤-2相关蛋白(Bax)和B淋巴细胞瘤-2(Bcl-2)]的表达情况;测量肿瘤大小为宽和长度,计算肿瘤大小;统计不同处理组肿瘤形成抑制率和总有效率。结果wIDH1组(100.37±10.24),对照组(102.45±13.57)和mIDH1组(100.63±12.42)之间细胞增殖差异无统计学意义(P>0.05)。mIDH1组U87细胞在CCK-8测定中显示出对替莫唑胺的敏感性,mIDH1组(36.84±3.55)细胞增殖和细胞活力较wIDH1组(98.17±8.54)和对照组(73.26±5.37)低(P<0.05);mIDH1组(62.18±3.47)%细胞的凋亡率高于对照组(33.16±6.54)%和wIDH1组(15.36±2.33)%;蛋白质免疫印迹和酶联免疫吸附测定(ELISA)结果显示,三组不同处理中,mIDH1组在Caspase-3,Caspase-9和Bax中的蛋白表达水平最高,Bcl-2的蛋白表达水平最低(P<0.05),wIDH1的异位过表达诱导Bcl-2蛋白表达水平的上调及Caspase-3,Caspase-9和Bax蛋白水平的下调(P<0.05);与其余两组相比,mIDH1组(220.17±12.15)mm^(3)的肿瘤体积最小(P<0.05)。wIDH1组(624.36±33.54)mm^(3)与对照组(601.17±36.18)mm^(3)差异无统计学意义(P>0.05);在替莫唑胺治疗组中,mIDH1组的总有效率为(73.15±8.46)%,wIDH1组的总有效率为(22.16±3.96)%。对照组的结果为(42.25±3.17)%。结论mIDH1通过上调替莫唑胺后胶质瘤细胞Caspase-3、Caspase-9、Bax表达并下调Bcl-2表达提高胶质瘤细胞凋亡率,增强治疗敏感性。

Objective To study the effect of isocitrate dehydrogenase 1(IDH1)gene mutation on the apoptosis of glioma U87 cells treated with temozolomide,in order to understand its biological characteristics and chemosensitivity.Methods The eukaryotic expression vector carrying the wild IDH1 gene(wIDH1)or the mutant IDH1 gene(mIDH1)was constructed by DNA recombination technology.The proliferation ability and cell viability of three stable cell lines were detected by CCK-8.Flow cytometry was used to detect the apoptosis rate of stable cell lines and the change in cell apoptosis rate after the intervention of temozolomide.The nude mice xenograft model was constructed and temozolomide was adopted for intervention.ELISA and Western blotting were used to detect the expressions of cellassociated apoptosis proteins(Caspase-3,Caspase-9,Bax and Bcl-2).The tumor size was measured as width and length,and the tumor size was calculated.The tumor formation inhibition rate and the total effective rate of different treatment groups were figured out.Results There was no significant difference in cell proliferation among wIDH1 group(100.37±10.24),control group(102.45±13.57)and mIDHl group(100.63±12.42)(P>0.05).U87 cells in mIDHl group showed sensitivity to temozolomide in CCK-8 assay.Cell proliferation and cell viability in mIDH1 group(36.84±3.55)were lower than those in wIDH1 group(98.17±8.54)and control group(73.26±5.37)(P<0.05).The apoptotic rate of cells in mIDH1 group(62.18±3.47)%was higher than that in control group(33.16±6.54)%and wIDH1 group(15.36±2.33)%.The protein immunoblotting and enzyme-linked immunosorbent assay(ELISA)results showed that among the three different treatments,the protein expression levels of Caspase-3,Caspase-9 and Bax were the highest in mIDH1 group,while the protein expression level of Bcl-2 was the lowest in mIDH1 group(P<0.05).The ectopic overexpression of wIDH1 induced the upregulation of Bcl-2 protein expression and the downregulation of Caspase-3,Caspase-9 and Bax protein expresssions(P<0.05).Compared with the other two groups,the mIDH1 group(220.17±12.15)mm^(3) had the smallest tumor volume(P<0.05).There was no significant difference in tumor volume between wIDH1 group(624.36±33.54)mm^(3) and control group(601.17±36.18)mm^(3)(P>0.05).In temozolomide treatment group,the total effective rate of mIDH1 group was(73.15±8.46)%,and the total effective rate of wIDH1 group was(22.16±3.96)%.The result of the control group was(42.25±3.17)%.Conclusion mIDH1 can increase the apoptosis rate and the sensitivity of glioma cells by up-regulating the expressions of Caspase-3,Caspase-9 and Bax in glioma cells and down-regulating the expression of Bcl-2.