DNMT1 通过 DNA 甲基化调控 miR-20a/TXNIP 介导的视网膜色素上皮细胞焦亡。
DNMT1 regulates miR-20a/TXNIP-mediated pyroptosis of retinal pigment epithelial cells through DNA methylation.
机构信息
Department of Ophthalmology, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032, Yunnan, China.
Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, 200031, China.
出版信息
Mol Cell Endocrinol. 2023 Nov 1;577:112012. doi: 10.1016/j.mce.2023.112012. Epub 2023 Jul 26.
BACKGROUND
Pyroptosis of retinal pigment epithelium (RPE) cells is associated with the etiology of diabetic retinopathy (DR). In this study, we investigated the effect of DNMT1 on RPE cell pyroptosis by regulating miR-20a/TXNIP expression through DNA methylation.
METHODS
High glucose (HG)-induced ARPE-19 cells and mice were injected with streptozotocin (STZ) to generate DR cells and animal models. RT‒qPCR was used to detect the expression of miR-20a, and methylation-specific PCR (MS-PCR) was used to determine the occurrence of methylation of miR-20a. The expression of pyroptosis-related proteins (caspase-1 and NLRP3) and DNA methyltransferase (DNMT1) was detected by western blotting, and the expression of inflammatory factors (IL-1β and IL-18) was detected by ELISA. Apoptosis was detected by flow cytometry and TUNEL. HE staining was used to observe the pathological changes in retinal tissue in mice.
RESULTS
In HG-induced DR cell models, the expression of miR-20a was significantly downregulated, while the expression of inflammatory factors (IL-1β, IL-18) and pyroptosis-associated proteins (caspase-1, NLRP3) was significantly upregulated. Transfection of miR-20a mimic can effectively reverse HG-induced pyroptosis and release of inflammatory factors. DNMT1 promotes miR-20a methylation and inhibits the expression of miR-20a. DNMT1-mediated methylation is involved in the pyroptosis process of high glucose-induced RPE cells, and silencing DNMT1 can promote the expression of miR-20a, thereby inhibiting the release of IL-1β and IL-18 and reducing the occurrence of cell pyroptosis. miR-20a targets negative regulation of TXNIP expression, and overexpression of TXNIP can effectively reverse the inhibitory effect of miR-20a on pyroptosis. The methylation inhibitor 5-AZ can inhibit the occurrence of pyroptosis and DR processes, while treatment with a miR-20a inhibitor or OE-TXNIP can reverse the effect of 5-AZ.
CONCLUSION
DNMT1 promotes DNA methylation, decreases the expression of miR-20a and increases the expression of TXNIP, which ultimately leads to the occurrence of pyroptosis in RPE cells.
背景
视网膜色素上皮（RPE）细胞的细胞焦亡与糖尿病视网膜病变（DR）的病因有关。在这项研究中，我们通过 DNA 甲基化调节 miR-20a/TXNIP 的表达来研究 DNMT1 对 RPE 细胞焦亡的影响。
方法
用高糖（HG）诱导 ARPE-19 细胞和小鼠，并用链脲佐菌素（STZ）注射生成 DR 细胞和动物模型。用 RT‒qPCR 检测 miR-20a 的表达，用甲基化特异性 PCR（MS-PCR）检测 miR-20a 发生甲基化的情况。用 Western blot 检测细胞焦亡相关蛋白（caspase-1 和 NLRP3）和 DNA 甲基转移酶（DNMT1）的表达，用 ELISA 检测炎症因子（IL-1β 和 IL-18）的表达。用流式细胞术和 TUNEL 检测细胞凋亡。用 HE 染色观察小鼠视网膜组织的病理变化。
结果
在 HG 诱导的 DR 细胞模型中，miR-20a 的表达明显下调，而炎症因子（IL-1β、IL-18）和细胞焦亡相关蛋白（caspase-1、NLRP3）的表达明显上调。转染 miR-20a 模拟物可以有效逆转 HG 诱导的细胞焦亡和炎症因子的释放。DNMT1 促进 miR-20a 甲基化，抑制 miR-20a 的表达。DNMT1 介导的甲基化参与了高糖诱导的 RPE 细胞焦亡过程，沉默 DNMT1 可以促进 miR-20a 的表达，从而抑制 IL-1β 和 IL-18 的释放，减少细胞焦亡的发生。miR-20a 负调控 TXNIP 的表达，过表达 TXNIP 可以有效逆转 miR-20a 对细胞焦亡的抑制作用。甲基化抑制剂 5-AZ 可以抑制细胞焦亡和 DR 过程的发生，而用 miR-20a 抑制剂或 OE-TXNIP 处理可以逆转 5-AZ 的作用。
结论
DNMT1 促进 DNA 甲基化，降低 miR-20a 的表达，增加 TXNIP 的表达，最终导致 RPE 细胞发生细胞焦亡。
Zotero 插件