GPx1对肺癌A549微球体细胞增殖、凋亡的调控作用及可能的作用机制

The role of GPx1 in proliferation and apoptosis of lung cancer A549 spheres and its possible mechanism

目的:研究谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPx1)在调控肺腺癌A549微球体细胞增殖和凋亡中的作用,并探讨其可能的机制。方法:采用无血清悬浮法培养A549微球体细胞,应用FCM法检测A549微球体细胞中CD133^+CD44^+细胞所占的比例,蛋白质印迹法检测A549微球体细胞中干细胞标志物Sox2和Nanog蛋白的表达情况,以及GPx1蛋白的表达水平。采用质量浓度为5 mg/m L的顺铂(cisplatin,DDP)分别处理A549微球体细胞和亲本A549细胞,CCK-8法检测48 h内细胞的存活率;利用比色法检测A549微球体细胞和亲本A549细胞中谷胱甘肽(glutathione,GSH)的含量和GPx1的活性,蛋白质印迹法检测GPx1蛋白的表达情况;FCM法检测A549微球体细胞和亲本A549细胞中活性氧(reactive oxygen species,ROS)的水平。将特异性针对GPx1基因的GPx1-si RNA转入A549微球体细胞中,分别采用实时荧光定量PCR和蛋白质印迹法检测GPx1 m RNA及蛋白的表达水平;FCM法、蛋白质印迹法和细胞成球实验分别检测沉默GPx1基因表达后对A549微球体细胞中ROS水平、Sox2和Nanog蛋白表达水平以及A549微球体细胞成球能力的影响。应用FCM法、CCK-8法以及蛋白质印迹法检测转入GPx1-si RNA并联合DDP(5mg/m L)干预后,对细胞的生存率和凋亡水平以及磷酸化p38(phospho-p38,p-p38)、磷酸化激活转录因子2(phospho-activating transcription factor 2,p-ATF2)、p53和Bax蛋白表达水平的影响。结果:培养获得A549微球体细胞,A549微球体细胞中CD133+CD44+细胞所占比例为(11.7±0.6)%,明显高于亲本A549细胞的(2.2±0.3)%。Sox2和Nanog蛋白在A549微球体细胞中的表达水平明显高于在亲本A549细胞中的表达水平(P值均<0.05);DDP(5 mg/mL)处理48 h后,对A549微球体细胞的生存率无明显影响(P>0.05)。A549微球体细胞中GSH含量、GPx1的活性和蛋白的表达水平均明显高于亲本A549细胞(P值均<0.05),而ROS含量在A549微球体细胞中明显低于亲本A549细胞(P<0.05)。转入GPx1-siRNA可明显下调A549微球体细胞中GPx1 mRNA和蛋白的表达水平、并抑制Sox2蛋白的表达水平和微球体的形成(P值均<0.05)。下调GPx1表达并联合DDP处理后,A549微球体细胞的存活率明显下调(P<0.05),而细胞凋亡率明显升高(P<0.05)。此外,下调GPx1表达并联合DDP处理后,A549微球体细胞中p-p38、p-ATF2、p53和Bax蛋白的表达水平均明显上调(P值均<0.05)。结论:下调GPx1可能通过抑制Sox2、上调ROS和激活p38-p53通路发挥抑制细胞增殖、促进凋亡等作用,其可成为肺癌治疗的潜在靶点。

Objective： To investigate the effects of glutath apoptosis of lung adenocarcinoma A549 pheres in one peroxidase 1 （GPxl） on proliferation and vitro, and to explore its possible mechanism Methods： A549 spheres were cultured in serum-free medium. The proportion of CD133^＋CD44^＋ cells in A549 spheres was detected by FCM, and the expression levels of GPxl protein and stem cell markers Sox2 and Nanog were detected by Western blotting. The A549 spheres and their parental A549 cells were treated with 5 mg/mL cisplatin （DDP） for 48 h, then the cell survival rate was detected by CCK-8 method, the glutathione （GSH） concentrations and GPxl activities in A549 spheres and parental A549 cells were measured by colorimetric method, while the expression level of GPx1 protein and the level of reactive oxygen species （ROS） were detected by Western blotting and FCM, respectively. When GPx1-siRNA was transfected into A549 spheres, the expression levels of GPxl mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After silencing GPx1 gene expression in A549 spheres, the level of ROS in A549 spheres was detected by FCM, the expression levels of Sox2 and Nanog proteins were analyzed by Western blotting, and the sphere formation ability was detected by sphere-forming experiment. When A549 spheres were transfected with GPx1-siRNA and treated with DDP （5 mg/ mL）, the survival rate and the apoptosis rate of A549 sphere cells were measured by CCK-8 and FCM, respectively. Additional, the expression levels of phospho-p38 （p-p38）, phospho-activating transcription factor 2 （p-ATF2）, p53 and Bax proteins were detected by Western blotting. Results： A549 spheres were obtained successfully. The proportion of CD133^＋CD44^＋ cells was （11.7±0.6） % in all A549 spheres, which was higher than （2.2±0.3）% of parental A549 cells. The protein levels of Sox2 and Nanog in A549 spheres were higher than those in parental A549 cells （both P 〈 0.05）. After treatment with DDP （5 mg/mL） for 48 h, the survival rate of A549 spheres was not significantly changed （P 〉 0.05）. The GSH concentration, GPx1 activity and GPxl protein expression level in A549 spheres were higher than those in parental A549 cells （all P 〈 0.01）, but the ROS level in A549 spheres was lower than that in parental A549 cells （P 〈 0.05）. After GPx1 - siRNAs were transfected into A549 spheres, the expression levels of GPx1 and Sox2 were down- regulated, and the sphere formation was suppressed （all P 〈 0.05）. After GPx1 gene-silencing and DDP （5 mg/mL） treatment, the survival rate of A549 spheres was significantly decreased, and the apoptosis rate was elevated （both P 〈 0.05）, while the protein expression levels of p-p38, p-ATF2, p53 and Bax were significantly up-regulated （all P 〈 0.05）. Condusion： Down-regulation of GPx1 expression may suppress the expression of Sox2 and increase the level of ROS, so as to inhibit the proliferation and induce the apoptosis of A549 spheres via p38-p53 signal pathway. Thus GPxl may be a novel potential target for lung cancer treatment.