Knockdown of KCNQ1OT1 Alleviates the Activation of NLRP3 Inammasome 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 inammasome activation displays a crucial therapeutic value in DR. Studies have shown that KCNQ1OT1 plays a critical role in regulating NLRP3 inammasome 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 inammasome in DR. The expression of KCNQ1OT1 and the activation of NLRP3 inammasome were increased in experimental DR models. KCNQ1OT1 knockdown alleviated NLRP3 inammasome-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 inammasome through miR-17-5p/TXNIP axis. Moreover, the clinical samples of patients with DR showed that the expression of KCNQ1OT1 and the activation of NLRP3 inammasome were all increased, further supporting the hypothesis that the KCNQ1OT1 dysregulation may be the molecular mechanism of the pathogenesis of DR. Therefore, KCNQ1OT1 may serve as a new therapeutic target for DR. decreases NLRP3 activation by targeting miR-25-3p/phosphatase and tensin homolog (PTEN) /proteinserine/threonine kinase (PSKs) Western blot was performed to detect the protein level NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β, cleaved IL-1β in Müller molecules compared to loading GAPDH overexpression


Introduction
Diabetic retinopathy (DR), one of the most severe and common complications caused by diabetic mellites, can eventually lead to irreversibly visual impairment and is the leading cause of blindness in working-age populations [1]. Our previous study has found that pro-Epigallocatechin-3-gallate (EGCG) inhibited hyperglycemia-induced proliferation and pro-angiogenic cytokine production in Müller cells, which was mediated by inhibiting thioredoxin-interacting protein (TXNIP)/NLRP3 in ammasome axis, suggesting that NLRP3 in ammasome activation plays a vital role in the pathogenesis of DR.
TXNIP is an early response gene induced by hyperglycemia and diabetes [2], which highly induces and mediates the expression of in ammatory cytokine interleukin-1 beta (IL-1β) and glial brillary acidic protein in the retina of diabetic mice [3]. In addition, TXNIP has been found involved in the activation of NLRP3 in ammasome and the promotion of caspase-1 expression [4]. The N-terminal pyridine segment of NLRP3 serves as a scaffold that nucleated ASC to recruit pro-caspase-1 into the in ammasome sensing pathogen-associated molecular patterns and danger-associated molecular patterns. Then, the active caspase-1 can lead to the conversion of IL-1β precursors (pro-IL-1β) into mature IL-1β. We have reported that inhibiting the activation of NLRP3 in ammasome protected Müller cells from gliosis activation and probably inhibited the development of DR, however the underlying mechanisms remain unknown.
Long non-coding RNAs (LncRNAs) are non-coding RNAs with more than 200 nucleotides in length which have no or limited protein-coding potential. The abnormal expression of lncRNAs has been found involved in several biological processes [5]. As one of the most common lncRNAs, KCNQ1 overlapping transcript 1 (KCNQ1OT1) plays a crucial role in regulating NLRP3 in ammasome activation and is involved in the pathogenesis of diabetic complications. KCNQ1OT1 was highly expressed in cardiac tissue and high glucose (HG)-induced cardiomyocytes from diabetic patients and diabetic mice. Silencing of KCNQ1OT1 attenuated NLRP3 assembly and caspase-1 activation as well as IL-1β expression and maturation by targeting miR-214/caspase-1 axis [6,7]. Downregulation of KCNQ1OT1 inhibited HGinduced oxidative stress, in ammation, and NLRP3 activation in renal tubular epithelial cells by upregulating the expression of miR-506-3p [8]. Moreover, in diabetic corneal endothelial keratopathy, KCNQ1OT1 knockdown decreased the expression of NLRP3 and cleaved caspase-1 and IL-1β maturation [9]. However, no studies have focused on the ability of KCNQ1OT1 to regulate NLRP3 in ammasome activation in Müller cells during DR. Therefore, in the present study, we aimed to evaluate the role of KCNQ1OT1 in the activation of NLRP3 in ammasome and to explore its potential mechanism.

Mice model of DR
Eight-week-old male C57BL/6J mice (wild-type, WT) were obtained from the Laboratory Animal Center of Soochow University and were fed adaptively for one week. The DR model was induced by a high-fat diet and intraperitoneal injection of streptozotocin 50 mg/d for ve consecutive days. The control group was fed a normal diet and injected intraperitoneally with sodium citrate buffer. All mice were maintained under standard laboratory conditions within a 12-hour light-dark cycle. Mice with tail vein blood glucose greater than 16.7 mmol/L were diagnosed with diabetes and were selected for the subsequent studies. After eight weeks of successful modeling, the mice were subjected to the corresponding experiments. All animal experiments were approved by the Animal Research Ethics Committee of Soochow University and conformed to the Chinese National Standard.

Cell culture and treatment
Rat retinal Müller cells rMC-1 were obtained from the American Type Culture Collection (ATCC). The cells were cultured in DMEM medium (Gibco, USA) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) (Gibco), and 10% fetal bovine serum (FBS, Gibco). The cells were incubated in a humidi cation incubator with 5% CO 2 at 37℃. Müller cells were cultured in 5 mM (average glucose) or 30 mM D-glucose (high glucose) (Sigma, USA) for 24 h to stimulate the diabetic environment before or after the speci c experiments.

Intravitreal injection
Intravitreal injections were performed before the diabetes induction. After anesthesia, approximately 1µL of adeno-associated virus with KCNQ1OT1-shRNA or scrambled-shRNA was injected into the mouse vitreous cavity with a 33G needle. Two weeks later, a diabetic mice model was built as previously mentioned.

Quantitative real-time PCR (qRT-PCR)
TRIzol® reagent (Invitrogen, USA) was used to extract total RNA of the mice retina, the Müller cells, and the peripheral blood mononuclear cells (PBMCs). 1µg of total RNA was reverse transcribed into cDNA with Revert Aid First Strand cDNA Synthesis Kit (Thermo Scienti c, USA). qRT-PCR was conducted with the powerUP SYBR Green Master Mix (Thermo Fisher Scienti c, USA). qRT-PCR was also performed [10]. The primers used in this study is showed in the Supplemental table 1. The relative expression of genes was calculated by the 2 −ΔΔCT . The levels of KCNQ1OT1, TXNIP, NLRP3, ASC, Caspase1, and IL-1β were normalized to GAPDH while miR-17-5p was normalized to U6.

Western blot
The western blot analysis was conducted [10]. In brief, the cultured Müller cells and the mice retina were collected and lysed with protein lysate. BCA assay kit (Beyotime, China) was used to detect the concentration of the protein. Equivalent samples were separated by SDS-PAGE and then transferred to PVDF membranes. Then the membranes were hybridized with primary antibodies against NLRP3 (Abcam, USA), ASC (Santa Cruz Biotechnology, USA), Caspase1 (Abcam; for cleaved caspase-1 and pro-caspase-1), IL-1β (Abcam; cleaved IL-1β and pro-IL-1β), and TXNIP (Abcam) at 4°C overnight. After washing, secondary antibodies were incubated the next day. Image J was used to quantify the intensities of the bands and GAPDH was used as a loading control.

Enzyme-linked immunosorbent assay (ELISA)
Protein extraction was puri ed from the retina of mice and culture medium of Müller cells. The concentration of in ammatory cytokine IL-1β was detected by a commercial ELISA Kits (Jiancheng, China) according to the instructions of the manufacturers.

Immunohistochemical
Brie y, the eye sections of the mice were washed with phosphate-buffered saline (PBS) and then incubated with the primary antibodies against caspase-1 (Proteintech) overnight at 4℃. The primary antibodies were diluted in PBS and supplemented with 0.5% Triton X-100. After washing with PBS, the sections were incubated with biotinylated mouse anti-rabbit IgG (Proteintech) at room temperature. After 30 min, the slides were incubated with avidin-biotin-peroxidase complex using ABC kit. Then 3,3'diaminobenzidine tetrahydrochloride was used to observe the color reaction. After washed in water, all sections were counterstained with hematoxylin. The results were obtained with a microscope (Leica).

Fluorescence in Situ Hybridization (FISH)
The FISH assay was conducted [12]. Cy3-labeled probes KCNQ1OT1, 18S, and U6 were obtained from RiboBio (Guangzhou, China). After prehybridization, Müller cells were incubated with the probes overnight at 37℃. The cell nuclei were stained with DAPI and image results were obtained with a confocal microscope (SP8, Leica, Germany).

Luciferase reporter assay
Luciferase reporters containing wild-type (WT) or mutated KCNQ1OT1 and TXNIP were cloned to the downstream of the re y luciferase gene. The miR-17-5p mimic and the negative control were cotransfected into Müller cells respectively. The dual-luciferase reporter assay system (Promega, Madison, WI) was used to measure the luciferase activities according to the instructions of manufacturers.

Clinical Specimens
Patients with type 2 diabetic mellites or non-diabetic controls attending Lixiang Eye Hospital were recruited for this study from July 2020 to April 2021. Fluorescein fundus angiography was conducted to assess DR and the fundus photographs were taken by ultra wide eld digitalretinal image by Optos 200TX. According to the Diabetic Retinopathy Disease Severity Scale, diabetics were divided into three groups: non-obvious retinopathy (NDR), non-proliferative diabetic retinopathy (NPDR), and PDR [13].
Patients who had other diabetic complications, had received intravitreal treatments or photocoagulation in the rst three months, or had other ocular diseases (such as keratitis, uveitis and retinal detachment) were excluded. All experimental protocols in this study followed the guidelines of the Declaration of Helsinki and were approved by the Human Ethics Committee of Lixiang Eye Hospital, Soochow University.

PBMCs isolation
The peripheral blood was collected from the vein of patients in non-diabetic controls, NDR, NPDR, and PDR (n = 5/group). The fresh peripheral blood was collected in an anticoagulant centrifuge tube and then centrifuged at 1800g for 5 minutes to remove the supernatant. An equal amount of PBS was added to the tube and then mixed by inversion. In another 15 ml centrifuge tube, separation of lymphocyte from Ficoll peripheral blood at room temperature (Solarbio, China) was added in advance. Then the diluted blood was slowly added along the wall of the tube. After centrifugation for 20 min, the liquid in the centrifuge tube was strati ed, and the white lm in the middle was carefully absorbed by the pipettor and added into another 15ml centrifuge tube. PBS 2 ml was added, and the supernatant was removed by centrifuging at 1800 g for 5 min. The procedure was repeated three times to wash the PBMCs.

Statistical Analysis
All data are presented as Mean ± SEM. The experiments in this study were conducted in at least triplicates. Statistical analysis was performed by Student's t-test (2 groups comparisons) or one-way ANOVA followed by Tukey's multiple comparison post-test (multi-group comparisons). P < 0.05 was considered statistically signi cant.

Results
3.1. The promotion of KCNQ1OT1 on the activation of the NLRP3 in ammasome in the retinas of mice with DR To explore the effect of KCNQ1OT1 on DR, KCNQ1OT1 was knocked down by KCNQ1OT1-shRNA intravitreal injection in the STZ-induced DR model. The expression of KCNQ1OT1 was increased in the retinas of mice with DR and decreased by using KCNQ1OT1-shRNA, (Fig. 1A). Then, to identify the effect of KCNQ1OT1 on the activation of NLRP3 in ammasome, the protein levels of NLRP3 in ammasomerelated factors, including NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β and cleaved IL-1β were detected, which were increased in the retinas of mice with DR and inhibited by KCNQ1OT1 knockdown (Figs. 1B-1G). In addition, caspase-1 immunoreactivity was also increased in mice with DR which was suppressed by KCNQ1OT1 knockdown, indicating that KCNQ1OT1 probably be involved in NLRP3 in ammasome activation.

KCNQ1OT1 knockdown alleviates the activation of the NLRP3 in ammasome in HG-treated Müller cells
We then detected the expression of KCNQ1OT1 in vitro after HG-treated. The results indicated that KCNQ1OT1 was signi cantly increased in HG-treated Müller cells and decreased by KCNQ1OT1 siRNA transfection ( Fig. 2A). Moreover, consistent with the results of in vivo studies, the protein levels of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β and cleaved IL-1β were upregulated but each was inhibited by KCNQ1OT1 knockdown (Figs. 2B-2H).

KCNQ1OT1 promotes the activation of the NLRP3 in ammasome by acting as a ceRNA in Müller cells
To explore the potential mechanism of the lncRNA in Müller cells, a FISH assay was performed to explore the subcellular localization of KCNQ1OT1. The results suggested KCNQ1OT1 was mainly localized in the cytoplasm in Müller cells (Fig. 3A), indicating that KCNQ1OT1 probably function in the cytoplasm. Hence, it was speculated that KCNQ1OT1 acted as a miRNA sponge. With Starbase 3.0 database, it was found that miR-17-5P had two binding sites for KCNQ1OT1 (Fig. 3D). Firstly, the expression of miR-17-5P was decreased in the retinas of mice with DR and HG-treated Müller cells, contrary to the expression of KCNQ1OT1 (Figs. 3B and 3C). The luciferase reporter gene assay showed that miR-17-5p mimic reduced the luciferase activity in cells transfected with wild-type KCNQ1OT1 sequence compared with the NC group (miR-17-5p mimic negative control). Co-transfection of miR-17-5p mimic and mutant sequence on one of the two binding sites (mut-KCNQ1OT1-1 or mut-KCNQ1OT1-2) inhibited the luciferase activity. In contrast, miR-17-5p mimic stimulation had no effect on the luciferase activity elicited by the double mutations (mut-KCNQ1OT1-3) (Fig. 3D). To further investigate the effect of miR-17-5P in Müller cells, miR-17-5P mimic was co-transfected with pcDNA-KCNQ1OT1. We found that miR-17-5P mimic decreased the protein levels of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, pro-IL-1β and cleaved IL-1β, which was reversed by pcDNA-KCNQ1OT1 co-transfection (Figs. 3E and 3I).
3.4. KCNQ1OT1/miR-17-5P/TXNIP is involved in regulating the activation of NLRP3 in ammasome in Müller cells To verify the speci c mechanism of KCNQ1OT1 in regulating the activation of NLRP3 in ammasome in HG-treated Müller cells, Starbase 3.0 and Targetscan were used to predict the target gene of miR-17-5p. It should be noted that TXNIP have two binding sites with miR-17-5p (Fig. 4C) and is closely related to NLRP3 in ammasome. Firstly, the expression of TXNIP was increased in the retinas of mice with DR and HG treated Müller cells (Figs. 4A-4B). Then luciferase reporter gene assay was conducted to further veri ed the relationship between miR-17-5p and TXNIP. The cells were co-transfected with miR-17-5p mimic and luciferase vectors carrying the wild-type TXNIP sequence or mutant sequence on two binding sites (mutTXNIP-1, mutTXNIP-2, or mutTXNIP-3). As shown in Fig. 4C, miR-17-5p mimic signi cantly inhibited the luciferase activity of wild-type TXNIP, mutTXNIP-1, or mutTXNIP-2 and had no effect on the luciferase activities of mutTXNIP-3 transfection (Fig. 4C).

Clinical relevance of KCNQ1OT1 dysregulation in DR
We then investigated the clinical signi cance of KCNQ1OT1/miR-17-5P/TXNIP signaling. The PBMCs were collected from the veins of patients with non-diabetic controls, NDR, NPDR, and PDR (n = 5/group). Figure 1A showed the representative results of the fundus photographs in each group.
qRT-PCR showed that the expression of KCNQ1OT1 was increased in the PBMCs of patients with NPDR and PDR and its expression was correlated with disease severity (Fig. 5B). In contrast, miR-17-5P expression was downregulated in patients with NPDR and PDR (Fig. 5C). Additionally, the levels of NLRP3 in ammasome-related factors, including NLRP3, ASC, caspase-1, IL-1β were also upregulated, which further supported the hypothesis that the KCNQ1OT1 is in involved in the activation of NLRP3 in ammasome and that KCNQ1OT1 dysregulation may be the molecular mechanism of the pathogenesis of DR.

Discussion
LncRNAs have been reported to be involved in the pathogenesis of DR [14][15][16]. In the present study, the activation of NLRP3 in ammasome in Müller cells is con rmed to be pivotal in HG-induced sterile in ammation. KCNQ1OT1 is an essential starting point in the process of NLRP3 in ammasome activation and KCNQ1OT1 knockdown inhibited HG-induced the activation of NLRP3 in ammasome in Müller cells, which expands the understanding of the pathogenesis of DR. Furthermore, KCNQ1OT1 regulates the activation of NLRP3 in ammasome 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 con rmed 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 in ammasome 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 in ammasome activation of Müller cells in DR remains unknown. Our study represents the rst 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 in ammasome 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 in ammasome [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 in ammasome 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 ux, while TXNIP knockdown prevents mitochondrial fragmentation, mitophagic ux 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 in ammation under chronic hyperglycemia [3]. TXNIP knockout prevents the HG-induced mitochondrial damage and mitophagy in mice Müller cells. TXNIP is also signi cantly 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 in ammasome-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 in ammasome activation in Müller cells under HG condition [25]. However, the regulatory mechanism of TXNIP remains unclear. In the present study, we nd 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 in ammasome 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. . DR is now discussed as a probable chronic in ammatory disease and displays a crucial therapeutic value on inhibition of NLRP3 in ammasome activation in the treatment of DR. Our results reveals that HG promotes NLRP3 in ammasome assembly and facilitates the activation of caspase-1. Cleaved caspase-1 then matures the in ammatory cytokines IL-1β in retinal Müller cells. KCNQ1OT1 knockdown inhibits the NLRP3/caspase-1 in ammasome signaling, which is mediated by miR-17-5p/TXNIP axis. This study extends our understanding on the role of NLRP3 in ammasome 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 in ammasome 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 in ammasome 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. Consent for Publication. All authors have reviewed the manuscript and have given consent for publication.

Con icts of interest
The authors declare that there is no con ict of interest.      The Schematic diagram of the mechanism of KCNQ1OT1 in Müller cells. Under HG environment, the expression of KCNQ1OT1 is increased. KCNQ1OT1 then promotes the expression of TXNIP by acting as a