Abstract
Objective
This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells.
Methods
The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair.
Results
ADR could significantly inhibit ARPE-19 cell proliferation and induce caspasedependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein–protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure.
Conclusions
The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
摘要
目的
通过比较阿霉素(ADR)作用的视网膜色素上皮细胞(ARPE-19)和正常ARPE-19 间的差异表达基因和信号通路,探究ADR 治疗玻璃体视网膜增殖性疾病的潜在机制。
创新点
首次运用基因芯片技术通过比较转录组学分析探究ADR促进ARPE-19 细胞凋亡的机制。
方法
采用磺酰罗丹明B(sulforhodamine B,SRB)比色法和碘化丙啶(PI)单染结合流式细胞术检测ADR 对ARPE-19 细胞的增殖抑制作用;通过基因芯片技术筛选ADR 作用的ARPE-19 细胞(实 验组)和正常ARPE-19 细胞(对照组)间的差异表达基因和相关信号通路;用JC-1 染色结合流式细胞术和Bcl2/Bax 蛋白表达比率检测线粒体功 能;通过检测γ-H2AX、p-CHK1、 p-CHK2 等蛋 白表达量分析DNA 的损伤和修复。
结论
ADR 通过启动DNA 损伤反应,引起p53 信号通路依赖的线粒体功能失调并激活caspase 依赖的凋亡,最终导致ARPE-19 细胞死亡。
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Project supported by the Zhejiang Province Key Research and Development Program (No. 2015C03042), China
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Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells
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Lin, Yc., Shen, Zr., Song, Xh. et al. Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells. J. Zhejiang Univ. Sci. B 19, 895–909 (2018). https://doi.org/10.1631/jzus.B1800408
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DOI: https://doi.org/10.1631/jzus.B1800408