Department of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Hunan Clinical Research Center of Ophthalmic Disease, Changsha, Hunan 410011, China.
Invest Ophthalmol Vis Sci. 2021 Mar 1;62(3):13. doi: 10.1167/iovs.62.3.13.
Apoptosis of the retinal ganglion cells (RGCs) can cause irreversible damage to visual function after retinal ischemia reperfusion injury (RIR). Using a lncRNA chip assay, we selected lncRNA Ttc-209 and characterized its role in RGCs during ischemia reperfusion (I/R)-induced apoptosis.
We created an ischemic model of RGCs by applying Hank's balanced salt solution containing 10 µM antimycin A and 2 µM calcium ionophore for 2 hours. RIR was induced in mice by elevating the intraocular pressure to 120 mm Hg for 1 hour by cannulation of the cornea; this was followed by reperfusion. Real-time quantitative PCR was used to detect the expression levels of long noncoding RNA (lncRNA), microRNA (miRNA), and target gene mRNA. Western blotting, flow cytometry, immunofluorescent staining, and TUNEL assays were performed to detect cell apoptosis. Dual-luciferase reporter assays and FISH were used to identify endogenous competitive RNA (ceRNA) mechanisms that link lncRNAs, miRNAs, and target genes. We also used scotopic electroretinography examinations to evaluate visual function in treated mice.
lncRNA Ttc3-209 was significantly upregulated after I/R injury and played a proapoptotic role in RGCs during I/R-induced apoptosis. Mechanistically, lncRNA Ttc3-209 is a ceRNA that competitively binds to miR-484 and upregulates the translation of its target (Wnt8a mRNA), thus promoting apoptosis in RGCs.
Reducing the expression of lncRNA Ttc3-209 had a protective effect against apoptosis in RGCs. This may provide a new therapeutic option for the prevention of RGC apoptosis in response to RIR injury.
视网膜神经节细胞（RGCs）的凋亡可导致视网膜缺血再灌注损伤（RIR）后视觉功能的不可逆损害。本研究通过 lncRNA 芯片检测，选择 lncRNA Ttc-209，并对其在缺血再灌注（I/R）诱导的 RGC 凋亡中的作用进行鉴定。
通过在 Hank's 平衡盐溶液中添加 10 μM 抗霉素 A 和 2 μM 钙离子载体孵育 2 小时，建立 RGC 缺血模型。通过角膜套管将眼内压升高至 120mmHg 持续 1 小时，诱导 RIR；随后进行再灌注。实时定量 PCR 检测长非编码 RNA（lncRNA）、微小 RNA（miRNA）和靶基因 mRNA 的表达水平。通过 Western blot、流式细胞术、免疫荧光染色和 TUNEL 检测分析细胞凋亡。双荧光素酶报告基因检测和 FISH 鉴定 lncRNA、miRNA 和靶基因之间的内源性竞争性 RNA（ceRNA）机制。此外，还通过暗适应视网膜电图检查评估处理后小鼠的视觉功能。
I/R 损伤后 lncRNA Ttc3-209 显著上调，在 I/R 诱导的 RGC 凋亡中发挥促凋亡作用。机制上，lncRNA Ttc3-209 作为 ceRNA，与 miR-484 竞争结合并上调其靶基因（Wnt8a mRNA）的翻译，从而促进 RGC 凋亡。
降低 lncRNA Ttc3-209 的表达对 RGC 凋亡具有保护作用。这可能为预防 RIR 损伤引起的 RGC 凋亡提供新的治疗选择。
