非小细胞肺癌患者血浆氧化应激水平和Klotho启动子甲基化分析

Level of oxidative stress and Klotho promoter methylation in the plasma of patients with non-small cell lung cancer

目的:探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者血浆氧化应激损伤水平和Klotho蛋白启动子甲基化状态及临床意义。方法:选择66名在四川省人民医院确诊为NSCLC的患者,另选50名健康人群作为对照组,分别采集其血浆。血浆超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)和过氧化氢酶(catalase,CAT)水平采用试剂盒检测。qPCR和Western blot检测血浆Klotho的表达。Klotho表达量与氧化应激的相关性采用Pearson相关性分析。甲基化特异性PCR(MS-PCR)检测Klotho甲基化水平。CpG岛甲基化率采用焦磷酸盐测序法。结果:相对于对照组[(94.56±16.40)m U/L],观察组SOD含量[(79.86±18.24)mU/L]明显降低(t=4.487,P=0.000),观察组MDA水平[(2.87±2.26)pg/mL]与对照组[(2.52±1.06)pg/mL]无统计学差异(t=1.675,P=0.097),观察组CAT水平[(18.50±4.62)U/mg]与对照组[(25.26±3.54)U/mg]相比明显降低(t=8.605,P=0.000)。观察组(0.66±0.16)比对照组(1.04±0.10)血浆Klotho基因(t=14.93,P=0.000)降低;观察组(0.70±0.20)比对照组(1.04±0.10)血浆Klotho蛋白表达(t=10.92,P=0.000)降低,且Klotho蛋白表达量与SOD水平(r=0.768,P=0.000)和CAT水平(r=0.708,P=0.000)呈正相关。观察组Klotho启动子CpG岛甲基化水平升高。结论:Klotho甲基化可能是NSCLC患者血浆氧化应激状态的一个临床标志物。

Objective:To investigate the level of oxidative stress and Klotho promoter methylation in the plasma of patients with nonsmall cell lung cancer(NSCLC)and their clinical significance. Methods:Plasma samples were collected from 66 patients,who were diagnosed with NSCLC in Sichuan Provincial People’s Hospital(as observation group),and 50 healthy people(as control group).Assay kits were used to determine the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and catalase(CAT)in plasma.The mRNA and protein expression of Klotho in plasma was measured/determined by qPCR and Western blot,respectively. Pearson correlation analysis was performed for the correlation between Klotho expression level and oxidative stress. Klotho methylation level was determined by methylation-specific PCR(MS-PCR). The methylation rate of CpG islands was determined by pyrosequencing.Results:Compared with the control group,the observation group had significantly reduced levels of SOD[(79.86±18.24)mU/L vs.(94.56±16.40)mU/L,t=4.487,P=0.000] and CAT[(18.50±4.62)U/mg vs.(25.26±3.54)U/mg,t=8.605,P=0.000],but there was no significant difference in MDA level between the observation group and the control group[(2.87±2.26) pg/mL vs.(2.52 ±1.06)pg/mL,t= 1.675,P=0.097]. The mRNA and protein expression levels of Klotho in plasma were significantly lower in the observation group than in the control group(mRNA:0.66±0.16 vs. 1.04±0.10,t=14.93,P=0.000;protein:0.70±0.20 vs. 1.04±0.10,t=10.92,P= 0.000). There was a positive correlation between the protein expression of Klotho and the levels of SOD(r=0.768,P=0.000)and CAT(r=0.708,P=0.000). Meanwhile,the observation group had an increased methylation level of CpG islands in the Klotho promoter. Conclusion :Klotho methylation may be a clinical marker of oxidative stress in the plasma of patients with NSCLC.