Prdx4蛋白表达水平的改变对人肺腺癌A549细胞增殖、凋亡和迁移的影响

发布时间：2018-02-28 08:15

本文关键词： 癌非小细胞肺过氧化物还原酶细胞增殖细胞凋亡细胞运动　出处：《肿瘤》2017年04期 　论文类型：期刊论文

【摘要】：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(P0.01);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(P0.05和P0.01);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(P0.05和P0.01)。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(P0.01);沉默Prdx4表达的A549细胞的愈合率明显低于对照组(P0.01)。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(P0.01);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(P0.01)。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。
[Abstract]:Objective: to investigate the effect of up-regulating or silencing the expression of peroxiredoxin 4prdx4) on the proliferation of human lung adenocarcinoma cell line A549. Methods: recombinant plasmid pcDNA3.0-HA-Prdx4 (empty vector pcDNA3.0-HA as control group) and siRNA-Prdx4-siRNA-specific targeting human Prdx4 gene (negative control group) were transfected into A549 cells by liposome transient transfection. The expression level of Prdx4 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blotting. The proliferation and apoptosis of A549 cells were detected by CCK-8 and immunofluorescence techniques respectively. Results: the expression level of Prdx4 mRNA and protein in A549 cells transferred into recombinant plasmid pcDNA3.0-HA-Prdx4 was significantly up-regulated by 0.05 P value, and Prdx4 mRNA and protein in Prdx4-siRNA A549 cells were detected by cell scratch assay and Transwell chamber assay. The results of CCK-8 assay showed that the D450 nm values of A549 cells with overexpression of Prdx4 were significantly higher than those of the control group at 24, 48 and 72 h, and the D450 nm values at 96 h were 2.241 卤0.068 and 1.596 卤0.103, respectively. The D450 nm values of A549 cells were significantly lower than those of the control group at 48, 72 and 96 h after the expression of Prdx4 was silenced. The results of immunofluorescence showed that the number of apoptotic cells in A549 cells with overexpression of Prdx4 was significantly lower than that in the control group at 24 and 48 h after stimulation with cisplatin. The difference was statistically significant (P 0.05 and P 0.01), and the expression of Prdx4 was silenced in A549 cells stimulated by cisplatin for 24 h and 48 h. The number of apoptotic cells was significantly higher than that of control group (P 0.05 and P 0.01). The results of scratch test showed that the healing rate of A549 cells with Prdx4 overexpression was significantly higher than that of control group (P 0.01), and the healing rate of A549 cells with silencing Prdx4 expression was significantly lower than that of control group (P 0.01). Transwell migration assay showed that the healing rate of A549 cells was significantly higher than that of control group. The number of cells passing through the ventricular membrane of A549 cells with overexpression of Prdx4 was significantly higher than that of the control group (P0.01A), and the number of cells passing through the membrane of A549 cells with silencing Prdx4 expression was significantly lower than that of the control group (P 0.01). Conclusion: the proliferation and migration of A549 cells can be promoted and the apoptosis of A549 cells can be inhibited by the overexpression of Prdx4. It may be a potential target for clinical treatment of lung adenocarcinoma.
【作者单位】：南方医科大学病理生理学教研室广东省蛋白质组学重点实验室;
【基金】：国家自然科学基金资助项目(编号:81171958)~~
【分类号】：R734.2

【参考文献】