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

发布时间：2018-05-22 13:08

本文选题：肺肿瘤 + 肿瘤干细胞　；参考：《肿瘤》2017年03期

【摘要】：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(P0.05)。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(P0.05)。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(P0.05),而细胞凋亡率明显升高(P0.05)。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。
[Abstract]:Aim: to investigate the role of glutathione peroxidase (1(glutathione peroxidase 1G x 1) in regulating the proliferation and apoptosis of lung adenocarcinoma A549 microsphere cells and its possible mechanism. Methods: A549 microspheres were cultured by serum-free suspension method. The percentage of CD133 ~ CD44 ~ cells in A549 microspheres was detected by FCM assay. The expression of Sox2 and Nanog protein in A549 microspheres was detected by Western blot. And the expression level of GPx1 protein. The survival rate of A549 microsphere cells and parent A549 cells was measured by CCK-8 method with 5 mg/m / L cisplatin DDP. The content of glutathione (GSH) and the activity of GPx1 in A549 microsphere cells and parent A549 cells were detected by colorimetry. The expression of GPx1 protein was detected by Western blot and the level of reactive oxygen speciesROSs in A549 microspheres and parent A549 cells was detected by FCM. The specific GPx1-si RNA targeting GPx1 gene was transferred into A549 microsphere cells. The expression level of GPx1 m RNA and protein were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. The effects of silencing GPx1 gene expression on the expression of ROS, Sox2 and Nanog protein in A549 microspheres were detected by Western blot and cell spheroidization respectively. FCM assay and Western blot were used to detect the effects of GPx1-si RNA and DDP(5mg/m L on cell survival and apoptosis, and the expression of phosphorylated p38phospho-p38 p-p38, phosphorylated activator 2(phospho-activating transcription factor _ 2, p-ATF _ 2, p53 and Bax protein. Results: the percentage of CD133 CD44 cells in cultured A549 microsphere cells was 11.7 卤0.6g, which was significantly higher than that in parental A549 cells (2.2 卤0.3)%.Sox2 and Nanog). After 48 h treatment, the expression level of P was 0.05 mg / mL, and the level of P was 0.05 mg / mL. after treatment with DDP (5 mg / mL) for 48 h, There was no significant effect on the survival rate of A549 microsphere cells. The activity of GSH and the expression of protein in A549 microspheres were significantly higher than those in the parent A549 cells (P < 0.05), but the ROS content in A549 microspheres was significantly lower than that in the parent A549 cells. The expression of GPx1 mRNA and protein in A549 microspheres was significantly down-regulated by GPx1-siRNA, and the expression of Sox2 protein and the formation of microspheres were inhibited by 0.05 P value. After down-regulation of GPx1 expression and combined with DDP treatment, the survival rate of A549 microspheres cells decreased significantly, while the apoptosis rate of A549 cells increased significantly. In addition, after down-regulation of GPx1 expression combined with DDP treatment, the expression levels of p-p38, p-ATF2, p53 and Bax proteins in A549 microspheres were significantly up-regulated (P < 0.05). Conclusion: down-regulation of GPx1 may play a role in inhibiting cell proliferation and promoting apoptosis by inhibiting Sox2, upregulating ROS and activating p38-p53 pathway, which may be a potential target for lung cancer therapy.
【作者单位】：南昌大学第二附属医院呼吸内科;江西省新钢中心医院呼吸内科;江西省井冈山大学附属医院呼吸科;
【分类号】：R734.2

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