抑制野生型p53诱导的蛋白磷酸酶1提高肺腺癌细胞对砷剂的敏感性  

Inhibition of WIP1 in A549 cells enhances the sensitivity of cells to arsenic treatment

作　　者：罗擎英[1] 谷仕艳[2] 张遵真[2] 

机构地区：[1]四川农业大学食品学院,雅安625014 [2]四川大学华西公共卫生学院

出　　处：《卫生研究》2017年第3期389-395,共7页Journal of Hygiene Research

基　　金：国家自然科学基金面上项目(No.81372945)

摘　　要：目的研究抑制野生型p53诱导的蛋白磷酸酶1(wild-type p53-induced phosphatase 1,WIP1)提高肺腺癌细胞对砷剂的敏感性及其分子机制。方法设计并合成WIP1编码基因的靶向siRNA,以Lipofectimient 2000转染入肺腺癌A549细胞,从而抑制WIP1蛋白的表达。运用Western-blot检测抑制WIP1对三氧化二砷(As_2O_3)诱导的P53磷酸化蛋白及其下游效应因子的影响;采用流式细胞术检测抑制WIP1后细胞凋亡情况;以噻唑蓝(MTT)法检测抑制WIP1后细胞的存活率。结果以siRNA技术成功将A549细胞中WIP1 mRNA和蛋白表达水平均抑制70%左右。相同浓度As_2O_3(5~40μmol/L)处理后,WIP1表达抑制组细胞中P53 ser15表达低于对照组,而P53 ser46表达显著高于对照组。此外,相同浓度As_2O_3(10~40μmol/L)处理后,WIP1表达抑制组中P53 ser15下游效应因子——P53上调凋亡诱导蛋白(P53 up-regulated modulator of apoptosis,PUMA)的表达水平显著低于对照组,而P53ser46下游效应因子——细胞周期调节蛋白P21的表达显著高于对照组。与之对应的是,相同剂量As_2O_3(5~40μmol/L)作用下,WIP1表达抑制组细胞的凋亡率和细胞死亡率均显著高于对照组。结论抑制肺腺癌细胞中WIP1的表达可通过促进P53磷酸化进程提高砷引发肺腺癌细胞凋亡的效率。Objective To explore if inhibiting the expression of wild-type p53- induced phosphatase 1 （WIP1）could enhance the sensitivity of A549 cells to arsenic. Methods To inhibit expression of WIP1, WIP1 siRNA was transferred into A549 cells by using Lipofectamine 2000. Then the protein expression levels of P53 phosphorylation proteins and their downstream effectors were detected by western-blot analysis. Cell apoptosis were assessed by AnnexinV-FITC stain assay. The sensitivity of transferred cells to arsenic was detected by using MTT assay. Results The mRNA and protein expression level of WIP1 were all decreased by 70% in A549 cells transferred with WIP1 siRNA.Western-blot analysis indicated that P53 phosphorylation process was much accelerated in WIPl-inhibited cells after arsenic treatment. For example, compared to control ceils, an significant decrease in P53 serl5 expression and an increase in P53 ser46 expression was found in WIPl-inhibited cells when treated with As2O3 （5 -40 μmol/L）. In addition, compared to control group, the expression of P21 decreased whereas PUMA increased in WIP1-inhibited cells when treated with As2O3 （10 -40 μmol/L）. Cell viability of WIP1-inhibited cells after As2O3 treatment （5 -40 μmol/L） was significantly higher than that of the control group, which may be due to a high apoptosis rate in WIP1- inhibited cells. Conclusion WIP1 could be used as a new target in arsenic-base anticancer therapies.

关 键 词：野生型p53诱导的蛋白磷酸酶1 三氧化二砷 凋亡 增敏 A549细胞 

分 类 号：R734.2[医药卫生—肿瘤] Q555.7[医药卫生—临床医学]