Department of Ophthalmolog, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515000, China.
Department of Ophthalmolog, The First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515000, China.
Biomed Pharmacother. 2018 Apr;100:349-357. doi: 10.1016/j.biopha.2018.02.001. Epub 2018 Feb 16.
This study aimed to investigate the effect and potential mechanism of miR-1298 in the progression of human trabecular meshwork (HTM) cells.
Expression of miR-1298 was assessed by quantitative real time PCR (qRT-PCR), as well as in HTM-1 and HTM-2 cells. Mature miR-1298 mimic, miR-1298 inhibitor, and si-EIF4E3 and their corresponding controls were transfected into HTM-1 and HTM-2 to obtain stable HTM cells. Luciferase reporter assay was used to verify regulation between miR-1298 and EIF4E3. Cytotoxicity and Oxidative damage were assessed using commercial kits, and apoptosis was determined using flow cytometry. ECM and apoptosis related factors were determined using qRT-PCR and western blotting, as well as the pathway related factors.
The expression of miR-1298 was significantly decreased both in glaucoma and HTM cells. MiR-1298 mimic could significantly inhibit the increase of cytotoxicity, apoptosis, accumulation of carbonylated proteins and ECM induced by COS, but miR-1298 inhibitor could obviously promote the increase effects caused by COS in HTM cells. EIF4E3 was a downstream target of miR-1298. Sliced EIF4E3 could significantly inhibit the increase effects induced miR-1298 inhibitor in HTM cells under COS. The expression levels of TGF-β2 and Smad4 were significantly increased, and Wnt3a and β-cantenin were obviously decreased under COS, and miR-1298 inhibitor could markedly promote this increase effect, while sliced EIF4E3 could reverse the effect of miR-1298 under COS.
miR-1298 could protect HTM cells to against damage caused by COS via inhibiting TGF-β2/Smad4 pathway and activating canonical Wnt pathway.
本研究旨在探讨 miR-1298 在人眼小梁细胞（HTM）进展中的作用及其潜在机制。
通过实时定量 PCR（qRT-PCR）评估 miR-1298 的表达，并在 HTM-1 和 HTM-2 细胞中进行评估。将成熟的 miR-1298 模拟物、miR-1298 抑制剂、si-EIF4E3 及其相应的对照转染到 HTM-1 和 HTM-2 中，以获得稳定的 HTM 细胞。荧光素酶报告实验用于验证 miR-1298 与 EIF4E3 之间的调控关系。使用商业试剂盒评估细胞毒性和氧化损伤，使用流式细胞术测定细胞凋亡。使用 qRT-PCR 和 Western blot 以及通路相关因子测定 ECM 和凋亡相关因子。
miR-1298 的表达在青光眼和 HTM 细胞中均显著降低。miR-1298 模拟物可显著抑制 COS 诱导的细胞毒性、凋亡、羰基化蛋白积累和 ECM 增加，但 miR-1298 抑制剂可明显促进 COS 引起的 HTM 细胞的增加作用。EIF4E3 是 miR-1298 的下游靶标。在 COS 条件下，切割的 EIF4E3 可显著抑制 miR-1298 抑制剂诱导的 HTM 细胞的增加作用。COS 条件下，TGF-β2 和 Smad4 的表达水平显著升高，Wnt3a 和 β-cantenin 明显降低，miR-1298 抑制剂可显著促进这种增加作用，而切割的 EIF4E3 可逆转 COS 下 miR-1298 的作用。
miR-1298 通过抑制 TGF-β2/Smad4 通路和激活经典 Wnt 通路，可保护 HTM 细胞免受 COS 引起的损伤。
