Aim Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is the key of the development of diabetic retinopathy (DR), and lncRNA NEAT1 could accelerate EMT in diabetic nephropathy. Meanwhile, as a diabetes susceptibility gene, whether sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) has relationship with lncRNA NEAT1 in DR remains unclear. Methods Firstly, NEAT1, SOX4 and miR-204 were evaluated by qRT-PCR (quantitative reverse-transcriptase PCR) under high glucose condition. Then, cell viability, proliferation, migration and invasion were respectively detected by MTT, BrdU staining, wound healing and transwell assay after NEAT1 knockdown or miR-204 overexpression. Also, the EMT-related proteins were examined by western blot and cell immunofluorescence assay. In order to confirm the relationship between miR-204 and NEAT1 or SOX4, dual luciferase reporter gene assay was conducted. At the same time, the protein levels of SOX4 and EMT-related proteins were investigated by immunohistochemistry in vivo. Results High glucose upregulated NEAT1 and SOX4 and downregulated miR-204 in ARPE19 cells. NEAT1 knockdown or miR-204 overexpression inhibited the proliferation and EMT progression of ARPE19 cells induced by high glucose. NEAT1 was identified as a molecular sponge of miR-204 to increase the level of SOX4. The effect of NEAT1 knockdown on the progression of EMT under high glucose condition in ARPE19 cells could be reversed by miR-204 inhibitor. Also, NEAT1 knockdown inhibited retinal EMT in diabetic mice. Conclusion NEAT1 regulated the development of EMT in DR through miR-204/SOX4 pathway, which could provide reference for clinical prevention and treatment.

中文翻译：

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LncRNA NEAT1通过调节miR-204/SOX4轴调节糖尿病视网膜上皮间质转化


目的视网膜色素上皮(RPE)细胞的上皮-间质转化(EMT)是糖尿病视网膜病变(DR)发生发展的关键，lncRNA NEAT1可加速糖尿病肾病的EMT。同时，作为糖尿病易感基因，性别决定区Y相关（SRY）高迁移率族蛋白4（SOX4）与DR中的lncRNA NEAT1是否存在关系仍不清楚。方法首先，在高糖条件下通过qRT-PCR（定量逆转录酶PCR）评估NEAT1、SOX4和miR-204。然后，分别通过MTT、BrdU染色、伤口愈合和Transwell实验检测NEAT1敲低或miR-204过表达后的细胞活力、增殖、迁移和侵袭。此外，还通过蛋白质印迹和细胞免疫荧光测定法检查了 EMT 相关蛋白。为了确认miR-204与NEAT1或SOX4之间的关系，进行了双荧光素酶报告基因测定。同时，通过体内免疫组化研究SOX4和EMT相关蛋白的蛋白水平。结果 ARPE19 细胞中高糖上调 NEAT1 和 SOX4，下调 miR-204。 NEAT1敲低或miR-204过表达抑制高糖诱导的ARPE19细胞的增殖和EMT进展。 NEAT1 被鉴定为 miR-204 的分子海绵，可提高 SOX4 的水平。 miR-204抑制剂可以逆转NEAT1敲低对ARPE19细胞高糖条件下EMT进展的影响。此外，NEAT1 敲除可抑制糖尿病小鼠的视网膜 EMT。结论 NEAT1通过miR-204/SOX4通路调控DR中EMT的发生，为临床防治提供参考。