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目的:探讨上调或沉默过氧化物还原酶4(peroxiredoxin 4,Prdx4)蛋白表达对人肺腺癌A549细胞增殖、凋亡和迁移的影响。方法:利用脂质体瞬时转染法将重组质粒pcDNA3.0-HA-Prdx4(以空载体pcDNA3.0-HA为对照组)和特异性针对人Prdx 4基因的siRNA(Prdx4-siRNA)(以阴性对照-siRNA为对照组)分别转染A549细胞;采用实时荧光定量PCR和蛋白质印迹法分别检测Prdx4 mRNA和蛋白的表达水平;CCK-8法和免疫荧光技术分别检测A549细胞的增殖和凋亡情况;细胞划痕实验和Transwell小室法检测A549细胞迁移能力的改变。结果:转入重组质粒pcDNA3.0-HA-Prdx4的A549细胞中Prdx4 mRNA和蛋白的表达水平明显上调(P值均<0.05),转入Prdx4-siRNA的A549细胞中Prdx4 mRNA和蛋白的表达水平明显下调(P值均<0.05)。CCK-8检测结果显示,Prdx4过表达的A549细胞在24、48和72 h时的D 450 nm值均明显高于对照组(P值均<0.05),并且96 h时的D 450 nm值(2.241±0.068和1.596±0.103)差异更加明显(P<0.01);沉默Prdx4表达后A549细胞在48、72和96 h时的D 450 nm值明显低于对照组(P值均<0.01)。免疫荧光结果显示,Prdx4过表达的A549细胞在顺铂刺激24和48 h后凋亡细胞数与对照组相比明显减少,差异均具有统计学意义(P<0.05和P<0.01);沉默Prdx4表达的A549细胞在顺铂刺激24和48 h后,凋亡细胞数明显多于对照组(P<0.05和P<0.01)。划痕实验结果显示,Prdx4过表达A549细胞的愈合率明显高于对照组(P<0.01);沉默Prdx4表达的A549细胞的愈合率明显低于对照组(P<0.01)。Transwell迁移实验结果显示,Prdx4过表达的A549细胞穿过小室膜的细胞数明显多于对照组(P<0.01);沉默Prdx4表达的A549细胞穿过小室膜的细胞数明显少于对照组(P<0.01)。结论:Prdx4可促进A549细胞的增殖和迁移并抑制其凋亡,其有可能成为临床治疗肺腺癌的一个潜在靶点。
Objective: To investigate the effects of up-regulated or silenced peroxiredoxin 4 (Prdx4) protein on the proliferation, apoptosis and migration of human lung adenocarcinoma A549 cells. Methods: The recombinant plasmid pcDNA3.0-HA-Prdx4 (empty vector pcDNA3.0-HA as control) and siRNA specific for human Prdx 4 gene (Prdx4-siRNA) Negative control-siRNA control group) were transfected A549 cells; real-time fluorescent quantitative PCR and Western blot were used to detect Prdx4 mRNA and protein expression levels; CCK-8 method and immunofluorescence were detected A549 cell proliferation and apoptosis Cell scratch assay and Transwell chamber assay were used to detect the migration of A549 cells. Results: The expression of Prdx4 mRNA and protein was significantly up-regulated in A549 cells transfected with recombinant plasmid pcDNA3.0-HA-Prdx4 (all P <0.05). Prdx4 mRNA and protein expression in A549 cells transfected with Prdx4-siRNA Significantly decreased (P <0.05). The results of CCK-8 showed that the D 450 nm of Prdx4 overexpressing A549 cells at 24, 48 and 72 h were significantly higher than that of the control group (all P <0.05), and the values of D 450 nm at 96 h 2.241 ± 0.068 and 1.596 ± 0.103) (P <0.01). The D 450 nm of A549 cells after silencing Prdx4 expression at 48, 72 and 96 h was significantly lower than that of the control group (all P <0.01). The results of immunofluorescence showed that the number of apoptotic cells in Prdx4-overexpressing A549 cells after 24 and 48 hours of cisplatin treatment were significantly decreased compared with the control group (P <0.05 and P <0.01), and the Prdx4 The number of apoptotic cells in A549 cells after cisplatin stimulation for 24 and 48 h was significantly higher than that of the control group (P <0.05 and P <0.01). Scratch test results showed that the healing rate of Prdx4 over-expressing A549 cells was significantly higher than that of the control group (P <0.01). The healing rate of Prdx4-expressing A549 cells was significantly lower than that of the control group (P <0.01). The results of Transwell migration showed that the number of Prdx4-overexpressing A549 cells passing through the ventricular membrane was significantly higher than that of the control (P <0.01). The number of Prdx4-expressing A549 cells passing through the ventricular membrane was significantly less than that of the control <0.01). Conclusion: Prdx4 can promote the proliferation and migration of A549 cells and inhibit the apoptosis of A549 cells, which may become a potential target for clinical treatment of lung adenocarcinoma.