目的 探讨乳腺癌细胞中过氧化物还原酶4(PRDX4)的表达对增殖和迁移的影响及其机制.方法 从癌症基因组图谱(TCGA)和基因型-组织表达(GTEx)数据库中获取泛癌肿瘤样本和正常样本的PRDX4 mRNA数据和相应的临床数据.Wilcoxon检验分析PRDX4基因在肿瘤组织和正常组织中的表达差异;Kaplan-Meier法分析PRDX4与癌症患者生存率的关系;免疫组织化学染色分析PRDX4在常见肿瘤及其癌旁组织中的表达.2,7-二氯荧光素二乙酸酯(DCFHDA)荧光探针检测细胞内活性氧水平;MTS比色法检测细胞增殖活性;碘化丙啶染色流式细胞术检测细胞周期变化;划痕愈合实验检测细胞迁移能力;蛋白质印迹法检测增殖细胞核抗原(PCNA)、p-Rb、CyclinE1、CyclinD1、CDK2、CDK4、N-cadherin、Vimentin、基质金属蛋白酶9(MMP9)、ERK1/2和p-ERK1/2等相关蛋白表达水平.结果 人体多器官组织芯片结果显示,与癌旁组织(0.01±0.00)相比,乳腺癌组织(0.07±0.01)中PRDX4表达增加,差异有统计学意义,t=6.55,P=0.003.与si-NC组相比,si-PRDX4组上调细胞内活性氧水平(21.92±1.41 vs 31.30±1.41);抑制细胞增殖活性(1.45±0.05 vs0.85±0.01);下调PCNA(1.00±0.02 vs 0.74±0.03)蛋白表达;并将细胞阻滞在G0/G1期[(47.50±0.42)％vs(56.66±0.05)％];下调 p-Rb(1.00±0.01 vs 0.72±0.03)、CyclinE1(1.00±0.02 vs 0.62± 0.02)、CyclinD1(1.00±0.02 vs 0.70±0.02)、CDK2(1.00±0.02 vs 0.67±0.01)和 CDK4(1.00±0.01 vs 0.65± 0.01)蛋白表达.划痕愈合实验结果显示,与si-NC组[(48.79±0.39)％]细胞相比,si-PRDX4组[(21.33±3.14)％]细胞24 h后迁移率降低,差异有统计学意义,t=13.14,P＜0.001.蛋白质印迹结果显示,与si-NC组细胞相比,si-PRDX4组细胞 N-cadherin(1.00±0.03 vs 0.48±0.03)、Vimentin(1.02±0.03 vs 0.47±0.02)和 MMP9(1.01±0.03 vs 0.67± 0.03)表达量下降,差异有统计学意义,t值分别为21.73、16.90和13.71,P值分别为＜0.001、＜0.001和0.002.加入FR18024抑制ERK通路,与对照组相比,FR18024组细胞增殖相关蛋白PCNA(1.00±0.02 vs 0.74±0.03)表达下调,且迁移相关蛋白 N-cadherin(1.00±0.09 vs 0.67±0.04)、Vimentin(1.00±0.12 vs 0.77±0.04)和 MMP9(1.00± 0.11 vs 0.69±0.05)表达均下调,差异有统计学意义,t值分别为11.61、3.05、3.23和4.15,P值分别为＜0.001、0.038、0.032和0.014.结论 PRDX4可以通过ERK通路影响细胞增殖和迁移进而促进乳腺癌的恶性进展.
Analysis of the effect of PRDX4 on breast cancer through the ERK pathway
Objective To investigate the effects of peroxiredoxin 4(PRDX4)expression on proliferation and migration in breast cancer cells and its mechanism.Methods PRDX4 mRNA data and corresponding clinical data for pan-cancer tumor samples and normal samples were obtained from the The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)databases.The expression difference of PRDX4gene in tumor tissues and normal tissues was analyzed by Wilc-oxon test.Kaplan Meier curve was used to predict the relationship between PRDX4 expression and survival of cancer pa-tients.Immunohistochemical staining was used to analyze PRDX4 expression in common tumors and their paracancerous tissues.2',7'-Dichlorofluorescin diacetate(DCFHDA)fluorescent probe was used to detect intracellular reactive oxygen species levels,MTS colorimetric assay for cell proliferative activity,propidium iodide staining to detect cell cycle changes,scratch healing assay to detect cell migration ability.Western blot was used to detect protein levels of PCNA,p-Rb,cy-clinE1,cyclinD1,CDK2,CDK4,N-cadherin,Vimentin,MMP9,ERK1/2,and p-ERK1/2.Results Human multi-organ tis-sue microarray results showed that PRDX4 expression was increased in breast cancer tumor tissues(0.07±0.01)com-pared to paracancerous tissues(0.01±0.00),and the difference was statistically significant,t=6.55,P=0.003.Com-pared with the si-NC group,the si-PRDX4 group up-regulated the intracellular reactive oxygen species level(21.92±1.41 vs 31.30±1.41),inhibited cell proliferative activity(1.45±0.05 vs 0.85±0.01),and down-regulated PCNA(1.00± 0.02 vs 0.74±0.03)protein expression,blocked cells in G0/Gi phase[(47.50±0.42)％vs(56.66±0.05)％],and down-regulated p-Rb(1.00±0.01 vs 0.72±0.03),CyclinE1(1.00±0.02 vs 0.62±0.02),CyclinD1(1.00±0.02 vs 0.70±0.02),CDK2(1.00±0.02 vs 0.67±0.01),and CDK4(1.00±0.01 vs 0.65±0.01)protein expression.The re-sults of the scratch healing experiment showed that the migration rate of cells in the si-PRDX4 group[(21.33±3.14)％]reduced after 24 h compared with that of cells in the si-NC group[(48.79±0.39)％],and the difference was statistically significant,t=13.14,P＜0.001.The results of Western blot showed that the protein expressions of N-cadherin(1.00± 0.03 vs 0.48±0.03),Vimentin(1.02±0.03 vs 0.47±0.02)and MMP9(1.01±0.03 vs 0.67±0.03)were decreased in the si-PRDX4 group of cells compared with those of the si-NC group,and the differences were statistically significant,with t-values of 21.73,16.90 and 13.71 and P-values of＜0.001,＜0.001 and 0.002,respectively.When FR18024 was added to inhibit the ERK pathway,compared with the control group,the expression of cell proliferation-associated protein PCNA(1.00±0.02 vs 0.74±0.03)was down-regulated,and the expressions of migration-associated protein N-cadherin(1.00 ±0.09 vs 0.67±0.04),Vimentin(1.00±0.12 vs 0.77±0.04),MMP9(1.00±0.11 vs 0.69±0.05)were also down-regulated.The differences were statistically significant,with t-values of 11.61,3.05,3.23 and 4.15,respectively,P-values of＜0.001,0.038,0.032 and 0.014.Conclusion PRDX4 can affect cell proliferation and migration through the ERK pathway and thus promote the malignant progression of breast cancer.
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