Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
Department of Ophthalmology, The People's Hospital of Zhengzhou University, Zhengzhou, 450003, Henan, China.
Chem Biol Interact. 2019 Jun 1;306:39-46. doi: 10.1016/j.cbi.2019.03.021. Epub 2019 Mar 26.
MicroRNAs (miRNAs) have been regarded as potential modulators in varying ocular diseases, including age-related cataract (ARC). However, the roles of miR-221 in ARC progression and its underlying mechanism remain poorly understood. In this study, human lens epithelial cell line (SRA01/04) was used to investigate the potential function of miR-221 in vitro. The expressions of miR-221, sirtuin-1 (SIRT1) and E2F transcription factor 3 (E2F3) were measured in ARC tissues by quantitative real-time polymerase chain reaction and western blotting, respectively. To investigate the effect of miR-221, SIRT1 and E2F3 on cell apoptosis, SRA01/04 cells were transfected with miR-221 inhibitor, negative control inhibitor, pcDNA3.1-SIRT1 overexpression vector, pcDNA3.1-E2F3 overexpression vector or pcDNA3.1 empty vector. After the transfection, cell viability was detected in SRA01/04 cells at 0, 24, 48 or 72 h by cell counting kit-8 assay. Cell apoptosis was evaluated in transfected SRA01/04 cells by flow cytometry and western blotting at 72 h. The interaction between miR-221 and SIRT1 or E2F3 was probed by luciferase activity and RNA immunoprecipitation assays. Results showed that high expression of miR-221 was exhibited in ARC tissues compared with that in normal samples and associated with Lens Opacities Classification System III grades. Knockdown of miR-221 promoted cell viability and inhibited apoptosis in SRA01/04 cells. Moreover, both of SIRT1 and E2F3 levels were directly targeted by miR-221 and down-regulated in ARC tissues. Besides, overexpression of SIRT1 or E2F3 increased cell viability and suppressed apoptosis in SRA01/04 cells, which was reversed by addition of miR-221. We concluded that miR-221 promoted lens epithelial cells apoptosis through regulating SIRT1 and E2F3, providing a novel biomarker for treatment of ARC.
微小 RNA（miRNAs）已被认为是各种眼部疾病（包括年龄相关性白内障（ARC））的潜在调节剂。然而，miR-221 在 ARC 进展中的作用及其潜在机制仍知之甚少。在这项研究中，使用人晶状体上皮细胞系（SRA01/04）在体外研究 miR-221 的潜在功能。通过定量实时聚合酶链反应和 Western 印迹分别测量 ARC 组织中 miR-221、沉默调节蛋白 1（SIRT1）和 E2F 转录因子 3（E2F3）的表达。为了研究 miR-221、SIRT1 和 E2F3 对细胞凋亡的影响，将 miR-221 抑制剂、阴性对照抑制剂、pcDNA3.1-SIRT1 过表达载体、pcDNA3.1-E2F3 过表达载体或 pcDNA3.1 空载体转染到 SRA01/04 细胞中。转染后，通过细胞计数试剂盒-8 法分别在 0、24、48 和 72 h 检测 SRA01/04 细胞的细胞活力。72 h 时通过流式细胞术和 Western blot 评估转染 SRA01/04 细胞的细胞凋亡。通过荧光素酶活性和 RNA 免疫沉淀测定探测 miR-221 与 SIRT1 或 E2F3 之间的相互作用。结果表明，与正常样本相比，ARC 组织中 miR-221 表达较高，且与晶状体混浊分类系统 III 级相关。miR-221 敲低促进 SRA01/04 细胞的细胞活力并抑制细胞凋亡。此外，SIRT1 和 E2F3 的水平均直接受到 miR-221 的靶向调节，并在 ARC 组织中下调。此外，SIRT1 或 E2F3 的过表达增加了 SRA01/04 细胞的细胞活力并抑制了细胞凋亡，而添加 miR-221 则逆转了这种作用。我们得出结论，miR-221 通过调节 SIRT1 和 E2F3 促进晶状体上皮细胞凋亡，为 ARC 的治疗提供了新的生物标志物。
