Department of Ophthalmology, The Second Affiliated Hospital of Mudanjiang Medical University, Mudanjiang, China.
Department of Gerontology, The Second Affiliated Hospital of Mudanjiang Medical University, Mudanjiang, China.
J Cell Biochem. 2018 Jan;119(1):817-823. doi: 10.1002/jcb.26245. Epub 2017 Oct 5.
In this study, we characterized the functional role of long noncoding RNA (lncRNA), brain derived neurotrophic factor anti-sense (BDNF-AS) in regulating D-glucose-induced (DGI) apoptosis in human retinal pigment epithelial (RPE) cells. Human RPE cell line, ARPE-19 cells were cultured in vitro and treated with various concentrations of D-glucose for 24 h. A TUNEL assay was applied with immunohistochemical and quantitative approaches to assess the apoptotic effect of D-glucose. Under the condition of 50 mM D-glucose, qPCR was used to assess gene expression of BDNF and BDNF-AS in ARPE-19 cells. Using siRNA transfection, BDNF-AS was endogenously knocked down in ARPE-19 cells. The effects of BDNF-AS downregulation on DGI apoptosis and BDNF expression were assessed by TUNEL assay, qPCR, and Western blot, respectively. Furthermore, in BDNF-AS-downregulated ARPE-19 cells, secondary siRNA transfection was conducted to knock down endogenous BDNF expression. Its effect on BDNF-AS-associated apoptotic regulation was further evaluated. High concentrations of D-glucose induced significant apoptosis in ARPE-19 cells in vitro. With treatment of 50 mM D-glucose, BDNF was markedly downregulated whereas BDNF-AS upregulated in ARPE-19 cells. SiRNA-mediated BDNF-AS downregulation ameliorated DGI apoptosis and upregulated BDNF in ARPE-19 cells. In addition, inhibiting BDNF reversed the protective effect of BDNF-AS downregulation on DGI apoptosis. Our results suggest that BDNF-AS, through inverse regulation of BDNF, might play a critical role in the process of DGI apoptosis in diabetic retinopathy.
在这项研究中，我们描述了长非编码 RNA（lncRNA）脑源性神经营养因子反义（BDNF-AS）在调节人视网膜色素上皮（RPE）细胞中 D-葡萄糖诱导的（DGI）细胞凋亡中的功能作用。将人 RPE 细胞系 ARPE-19 细胞体外培养并以不同浓度的 D-葡萄糖处理 24 小时。通过 TUNEL 测定和免疫组织化学及定量方法评估 D-葡萄糖的凋亡作用。在 50mM D-葡萄糖的条件下，使用 qPCR 评估 ARPE-19 细胞中 BDNF 和 BDNF-AS 的基因表达。使用 siRNA 转染，在 ARPE-19 细胞中内源性敲低 BDNF-AS。通过 TUNEL 测定、qPCR 和 Western blot 分别评估 BDNF-AS 下调对 DGI 凋亡和 BDNF 表达的影响。此外，在 BDNF-AS 下调的 ARPE-19 细胞中，进行二次 siRNA 转染以敲低内源性 BDNF 表达。进一步评估其对 BDNF-AS 相关凋亡调节的影响。高浓度的 D-葡萄糖在体外诱导 ARPE-19 细胞发生明显凋亡。用 50mM D-葡萄糖处理时，BDNF 在 ARPE-19 细胞中明显下调，而 BDNF-AS 上调。siRNA 介导的 BDNF-AS 下调改善了 DGI 凋亡并上调了 ARPE-19 细胞中的 BDNF。此外，抑制 BDNF 逆转了 BDNF-AS 下调对 DGI 凋亡的保护作用。我们的结果表明，BDNF-AS 通过对 BDNF 的反向调节，可能在糖尿病视网膜病变中 DGI 凋亡过程中发挥关键作用。
