LncRNAs have been reported to be involved in the pathogenesis of DR[14–16]. In the present study, the activation of NLRP3 inflammasome in Müller cells is confirmed to be pivotal in HG-induced sterile inflammation. KCNQ1OT1 is an essential starting point in the process of NLRP3 inflammasome activation and KCNQ1OT1 knockdown inhibited HG-induced the activation of NLRP3 inflammasome in Müller cells, which expands the understanding of the pathogenesis of DR. Furthermore, KCNQ1OT1 regulates the activation of NLRP3 inflammasome via miR-17-5p/TXNIP axis in vitro and in vivo, indicating the potential of KCNQ1OT1 for the prevention and reversal of DR.
Recent studies have confirmed that KCNQ1OT1 plays a vital role in diabetic complications, such as diabetic cardiomyopathy[6], diabetic nephropathy[8], and diabetic ocular disease[9, 17]. KCNQ1OT1 and NLRP3 inflammasome are triggered in the corneal endothelium of diabetic patients and mice, as well as in HG-treated corneal endothelial cells. KCNQ1OT1 regulates the expression of caspase-1 and IL-1β by acting as a ceRNA and competitively binding miR-214 in corneal endothelial cells[9]. The expression of KCNQ1OT1 is elevated in HG-treated human retinal endothelial cells (hRECs) and in the aqueous humor of patients with DR compared with the normal control group. KCNQ1OT1 promotes DR progression by the regulation of miR-1470 and epidermal growth factor receptor (EGFR)[17]. However, whether KCNQ1OT1 is involved in regulating NLRP3 inflammasome activation of Müller cells in DR remains unknown. Our study represents the first report involving the expression and function of KCNQ1OT1 in diabetic retinal Müller cells. In the present study, we found that KCNQ1OT1 is highly expressed in the retinas of diabetic mice, HG-treated Müller cells and the PBMCs of patients with DR. And inhibiting KCNQ1OT1 ameliorates the activation of NLRP3 inflammasome both in vitro and in vivo, which is mediated via miR-17-5p/TXNIP axis. Thus, KCNQ1OT1 is expected to be an innovative therapeutic target for DR.
TXNIP, an endogenous inhibitor of thioredoxin (TRX), has been reported as an essential protein for activation of NLRP3 inflammasome[18]. TXNIP interacts with TRX and fails to activate NLRP3 in resting cells. Under oxidative stress, TXNIP is released from TRX and binds directly to the leucine-rich region of NLRP3, which results in inflammasome assembly [19]. Previous studies have revealed that TXNIP plays a crucial role in the pathological process of many retinal cells and is involved in the progression of DR[20]. TXNIP is one of the highest genes induced by HG and diabetes in retinal microvascular endothelial cells (RMECs). HG treatment increases the production of ROS and promotes pyroptotic cell death through TXNIP/NLRP3 axis[21, 22]. Therefore, HG increases the TXNIP expression at the mRNA and protein levels. The upregulation of TXNIP is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux, while TXNIP knockdown prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG environment.[23]. HG sustains TXNIP expression in the Müller glia of the retina in mice, and TXNIP is involved in Müller glia activation, ER stress, oxidative stress, and sterile inflammation under chronic hyperglycemia[3]. TXNIP knockout prevents the HG-induced mitochondrial damage and mitophagy in mice Müller cells. TXNIP is also significantly upregulated in the retina of diabetic mice in vivo and induced the expression of GFAP and LC3BII puncta, which are reversed by injection intravitreally of TXNIP siRNA [24]. We have reported previously that the protein levels of NLRP3 inflammasome-related molecules (including TXNIP, NLRP3, ASC, cleaved caspase-1, pro-caspase-1, cleaved IL-1β, and pro-IL-1β) are upregulated by HG treatment and downregulated by TXNIP knockdown. The interaction between TXNIP and NLRP3 was increased under HG condition, implying the crucial role of TXNIP in NLRP3 inflammasome activation in Müller cells under HG condition[25]. However, the regulatory mechanism of TXNIP remains unclear. In the present study, we find that the expression of TXNIP is increased in HG-stimulated Müller cells and in the retina of diabetic mice, which is consistent with previous studies. TXNIP plays a key role in promoting NLRP3 inflammasome activation in vitro and in vivo of DR models, which is regulated by KCNQ1OT1 through binding to miR-17-5pcompetitively. Furthermore, whether the effect of TXNIP in regulating oxidative stress, glial cell activation, endoplasmic reticulum stress and autophagy is regulated by KCNQ1OT1 remains to be further investigated.
NLRP3 inflammasome is compromised by NLRP3 associated with adapter protein, apoptosis-associated speck-like protein containing a caspase recruitment segment (ASC) and pro-caspase-1[26]. During NLRP3 inflammasome activation, the oligomerization of pro-caspase-1 proteins induces autoproteolytic cleavage to cleaved caspase-1. Cleaved caspase-1 cleaves pro-IL-1β to IL-1β [27, 28]. Studies have demonstrated that the activation of NLRP3 inflammasome plays a crucial role in the pathogenesis of DR[29]. Jinhua Gan et al. found that HG-induced the activation of NLRP3/caspase-1/GSDMD signaling pathway, which resulted in the loss of retinal pericytes in vitro model of DR[30]. In HG-treated human retinal pigment epithelium (RPE), overexpressed MELLT3 decreases NLRP3 inflammasome activation by targeting miR-25-3p/phosphatase and tensin homolog (PTEN) /proteinserine/threonine kinase (PSKs) axis[31]. DR is now discussed as a probable chronic inflammatory disease and displays a crucial therapeutic value on inhibition of NLRP3 inflammasome activation in the treatment of DR. Our results reveals that HG promotes NLRP3 inflammasome assembly and facilitates the activation of caspase-1. Cleaved caspase-1 then matures the inflammatory cytokines IL-1β in retinal Müller cells. KCNQ1OT1 knockdown inhibits the NLRP3/caspase-1 inflammasome signaling, which is mediated by miR-17-5p/TXNIP axis. This study extends our understanding on the role of NLRP3 inflammasome and KCNQ1OT1 in retinal Müller cells, providing potential biomarkers or therapeutic targets for DR.
In conclusion, it was found in this study that KCNQ1OT1 promoted the activation of NLRP3 inflammasome in vitro and in vivo of DR models, which was mediated by miR-17-5p/TXNIP axis. Moreover, in the clinical samples of patients with DR, the expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were all increased, further supporting the hypothesis that the KCNQ1OT1 dysregulation may be the molecular mechanism of the pathogenesis of DR. Therefore, KCNQ1OT1 might be an effective interference target for the prevention and treatment of DR.