Knockdown of KCNQ1OT1 Alleviates the Activation of NLRP3 Inflammasome Through miR-17-5p/TXNIP Axis in Retinal Müller Cells

Diabetic retinopathy (DR) is one of the most severe and common complications caused by diabetic mellites. Inhibiting NLRP3 inflammasome activation displays a crucial therapeutic value in DR. Studies have shown that KCNQ1OT1 plays a critical role in regulating NLRP3 inflammasome activation and participates in the pathogenesis of diabetic complications. The present study aims to explore the role, and the potential mechanism of KCNQ1OT1 in regulating the activation of NLRP3 inflammasome in DR. qRT-PCR was used to detect the expression of KCNQ1OT1, miR-17-5p, TXNIP, NLRP3, ASC, caspase-1 and IL-1β. Western blot was performed to detect the expression of NLRP3, ASC, caspase-1, IL-1β and TXNIP. Immunohistochemistry and immunostaining were performed to detect the expression of caspase-1. The levels of the inflammatory cytokine IL-1β were determined by ELISA assay. FISH was used to detect the subcellular localisation of KCNQ1OT1. Bioinformatic analysis, luciferase reporter assay and <i>in vitro</i> studies were performed to elucidate the mechanism of KCNQ1OT1-mediated dysfunction. The expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were increased in experimental DR models. KCNQ1OT1 knockdown alleviated NLRP3 inflammasome-associated molecules expression. In addition, KCNQ1OT1 was found to be localized mainly in the cytoplasm of Müller cells and facilitated TXNIP expression by acting as a miR-17-5p sponge. KCNQ1OT1 promoted the activation of NLRP3 inflammasome through miR-17-5p/TXNIP axis. In conclusion, it was found in this study that KCNQ1OT1 promoted the activation of NLRP3 inflammasome both <i>in vitro</i> and <i>in vivo</i>, which was mediated by miR-17-5p/TXNIP axis. KCNQ1OT1 might be an effective interference target for the prevention and treatment of DR.

糖尿病视网膜病变（Diabetic retinopathy, DR）是糖尿病（diabetic mellitus）引发的最为严重且常见的并发症之一。抑制NLRP3炎症小体（NLRP3 inflammasome）的活化在DR的治疗中具有关键的临床价值。已有研究证实，长链非编码RNA KCNQ1OT1在调控NLRP3炎症小体活化过程中发挥关键作用，并参与糖尿病并发症的发病机制。本研究旨在探讨KCNQ1OT1在DR中调控NLRP3炎症小体活化的作用及其潜在分子机制。本研究采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测KCNQ1OT1、miR-17-5p、TXNIP、NLRP3、ASC、半胱天冬氨酸蛋白酶-1（caspase-1）及白细胞介素-1β（IL-1β）的表达水平；采用蛋白质印迹法（Western blot）检测NLRP3、ASC、caspase-1、IL-1β与TXNIP的蛋白表达；采用免疫组织化学染色与免疫荧光染色检测caspase-1的表达情况；采用酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测炎性细胞因子IL-1β的水平；采用荧光原位杂交（fluorescence in situ hybridization, FISH）检测KCNQ1OT1的亚细胞定位。通过生物信息学分析、双荧光素酶报告基因实验及体外实验，阐明KCNQ1OT1介导的细胞功能异常的分子机制。实验结果显示，在实验性DR模型中，KCNQ1OT1的表达水平与NLRP3炎症小体的活化程度均显著升高；敲低KCNQ1OT1可有效缓解NLRP3炎症小体相关分子的异常表达。此外，KCNQ1OT1主要定位于视网膜Müller细胞的细胞质内，并通过作为miR-17-5p的分子海绵促进TXNIP的表达。KCNQ1OT1可通过miR-17-5p/TXNIP信号轴促进NLRP3炎症小体的活化。本研究最终证实，KCNQ1OT1在体内与体外环境中均可促进NLRP3炎症小体的活化，该调控作用由miR-17-5p/TXNIP信号轴所介导。综上，KCNQ1OT1有望成为预防与治疗DR的有效干预靶点。