October 2019
Volume 60, Issue 13
Open Access
Retina  |   October 2019
miR-590-3p Inhibits Pyroptosis in Diabetic Retinopathy by Targeting NLRP1 and Inactivating the NOX4 Signaling Pathway
Author Affiliations & Notes
  • Chufeng Gu
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
  • Deji Draga
    Department of Ophthalmology, Shigatse People's Hospital, Shigatse, Xizang, PR China
  • Chuandi Zhou
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
  • Tong Su
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
  • Chen Zou
    Eye Institute, Eye and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, PR China
  • Qing Gu
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
  • Tashi Lahm
    Department of Ophthalmology, Shigatse People's Hospital, Shigatse, Xizang, PR China
  • Zhi Zheng
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
  • Qinghua Qiu
    Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
    Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai, PR China
    Department of Ophthalmology, Shigatse People's Hospital, Shigatse, Xizang, PR China
  • Correspondence: Qinghua Qiu, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Hongkou District, Shanghai 200080, PR China; qinghuaqiu@163.com
  • Zhi Zheng, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Hongkou District, Shanghai 200080, PR China; zzheng88@sjtu.edu.cn
  • Footnotes
     CG, DD, and CZ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science October 2019, Vol.60, 4215-4223. doi:https://doi.org/10.1167/iovs.19-27825
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      Chufeng Gu, Deji Draga, Chuandi Zhou, Tong Su, Chen Zou, Qing Gu, Tashi Lahm, Zhi Zheng, Qinghua Qiu; miR-590-3p Inhibits Pyroptosis in Diabetic Retinopathy by Targeting NLRP1 and Inactivating the NOX4 Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2019;60(13):4215-4223. doi: https://doi.org/10.1167/iovs.19-27825.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To elucidate the mechanism whereby miR-590-3p regulates pyroptosis in diabetic retinopathy (DR).

Methods: Human retinal microvascular endothelial cells (HRMECs) incubated with high glucose (HG) were used to establish cell models, and the expression levels of miR-590-3p, caspase-1, IL-1β, NLRP1, NOX4, TXNIP, NLRP3, and ROS were determined. Additionally, miR-590-3p was altered using a mimic or an inhibitor, and siRNAs targeting NLRP1 and NOX4 were applied to explore the regulatory mechanism of miR-590-3p in DR. The relationships between miR-590-3p and NLRP1/NOX4 also were investigated using a luciferase reporter assay. Furthermore, vitreous tissue samples were collected to confirm pyroptosis in clinical DR.

Results: Downregulated miR-590-3p and upregulated NLRP1/NOX4 levels were observed in a cell culture model of DR. Inhibiting miR-590-3p upregulated NLRP1, the NOX4/ROS/TXNIP/NLRP3 pathway, and caspase-1. NLRP1 and NOX4 were confirmed as direct target genes of miR-590-3p. The overexpression of miR-590-3p or knockdown of NLRP1 and NOX4 increased cell activity and suppressed pyroptosis. Intriguingly, the upregulation of IL-1β induced the downregulation of miR-590-3p by lowering the DNA promoter activity of pri-miR-590.

Conclusions: HG induced pyroptosis in a cell culture model of DR, and the downregulation of miR-590-3p promoted pyroptotic death by targeting NLRP1 and activating the NOX4/ROS/TXNIP/NLRP3 pathway via an IL-1β–mediated positive feedback loop.

Diabetic retinopathy (DR) is a highly specific microvascular complication of diabetes mellitus (DM) and is the leading cause of blindness among working-age adults.1,2 Accumulating evidence suggests that inflammation is a crucial factor in the development of DR.3,4 Pyroptosis is a novel inflammatory form of regulated cell death, and unlike apoptosis and necrosis, is always driven by caspase-1.57 Nod-like receptors, especially NLR family pyrin domain containing 1 (NLRP1) and NLRP3, have important roles in the activation of caspase-1 and the subsequent processing of IL-1β and IL-18.8 A previous study showed that silencing NLRP1 attenuated pyroptosis in Alzheimer's disease.9 Furthermore, NLRP1 and NLRP3 promoted pyroptotic death in the setting of autoimmune thyroiditis.10 However, the molecular mechanisms underlying the effects of NLRP1 and NLRP3 on pyroptosis in DR remain unclear. 
A previous study by our group showed that hyperglycemia contributed to increased retinal vascular permeability and apoptosis in DR via the reactive oxygen species (ROS)/ thioredoxin-interacting protein (TXNIP)/NLRP3 axis.11 Additionally, NADPH oxidase 4 (NOX4) is a ROS-generating enzyme that participates in inflammatory processes, mainly through the NOX4/ROS pathway.12,13 In human skin keratinocytes and mouse skin, the production of inflammatory cytokines increases via the NOX4/ROS signaling pathway.12 Moreover, NOX4 aggravates nephrotoxicity by activating ROS-mediated programmed cell death.13 Thus, we hypothesized that NOX4 might regulate NLRP3-dependent pyroptosis via the ROS/TXNIP axis and, therefore, we aimed to further investigate the mechanism of the role of NOX4 in DR. 
MicroRNAs (miRNAs) are small endogenous noncoding RNAs comprising 21 to 23 nucleotides that regulate gene expression primarily by binding to the 3′-untranslated region (3′-UTR) of target mRNAs.14 Studies have indicated that miRNAs have critical roles in the pathogenesis of DR by mediating cell proliferation, migration, and death.15 miR-590-3p has been shown to promote proliferation in ischemia-reperfusion injury by inhibiting the NF-κB pathway,16 which has an active role in inflammatory responses.17 Moreover, miR-590-3p is associated with the accumulation of IL-18 in osteoblasts18 and bioinformatic analysis predicted that it binds to the 3′-UTR of NLRP1 and NOX4 (miRDB; available in the public domain at http://mirdb.org/). These results suggest that miR-590-3p is an inflammation-related miRNA. However, the role of miR-590-3p in pyroptosis contributing to DR remains unclear. Thus, we aimed to determine the function of miR-590-3p in regulating pyroptosis in DR by targeting NLRP1 and NOX4
Materials and Methods
Chemicals and Reagents
The following reagents were obtained from Invitrogen (Carlsbad, CA, USA): M199 tissue culture medium, fetal bovine serum, streptomycin/penicillin, and TRIzol reagent. Human fibroblast growth factor-basic (FGF-basic) was obtained from PeproTech (Rocky Hill, NJ, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). PrimeScript RT Master Mix (RR036A), TB Green Premix Ex Taq (RR420A), and the miRNA First-Strand Synthesis and SYBR kit (638315) were purchased from TaKaRa (Kumamoto, Japan). The Dual-Luciferase Reporter Assay System was obtained from Promega (Madison, WI, USA). The Caspase 1 Assay Kit (ab39412), IL-1β ELISA kit (ab46052), anti-caspase-1 antibody (ab1872), and anti-IL-1β antibody (ab9722) were purchased from Abcam (Cambridge, UK). Anti-NLRP1, anti-NOX4, anti-TXNIP, and anti-NLRP3 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-Factor VIII and anti-β-actin antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA). 
Cell Culture and Transfection
Human retinal microvascular endothelial cells (HRMECs) were purchased from Cell Systems Corporations (catalog no. ACBRI 181; Kirkland, WA, USA)19,20 and cultured in M199 medium supplemented with 20% fetal bovine serum, 3 ng/mL FGF-basic, 10 units/mL heparin, and 1% streptomycin/penicillin at 37°C under 5% CO2, 95% air. D-glucose was added to the medium at a final concentration of 30 mmol/L to generate high-glucose (HG) conditions. D-mannitol was used to adjust the osmotic pressure. 
The human miR-590-3p mimic, negative control (NC) mimic, miR-590-3p inhibitor, NC inhibitor, and small-interfering RNAs (siRNAs) targeting NLRP1 and NOX4 were purchased from GenePharma (Shanghai, China). The NOX4 expression plasmid (pcDNA3.1-hNOX4) was obtained from GeneChem (Shanghai, China). The cells were transfected according to the manufacturer's instructions. The oligonucleotide sequences are listed in Supplementary Table S1
Cell Viability Assay
A CCK-8 assay was performed to evaluate the viability of HRMECs. The cells were seeded onto 96-well plates (3650; Corning, NY, USA) and incubated with HG. CCK-8 solution (10 μL) was added to each well, and the cells were further incubated for 2 hours at 37°C. Absorbance measurements were taken at 450 nm. Cell viability was calculated by comparing the optical density (OD) of treated and untreated cells. 
RNA Extraction and Real-Time Quantitative PCR
Total RNA was extracted from HRMECs using TRIzol reagent and reverse transcribed into complementary DNA using PrimeScript RT Master Mix or miRNA First-Strand Synthesis kits, following the manufacturer's protocols. Real-time quantitative PCR (qRT-PCR) was performed using TB Green Premix Ex Taq. The oligonucleotide primers used for PCR amplifications were purchased from BioSune Biotechnology (Shanghai, China) and are listed in Supplementary Table S2
Western Blotting
Proteins extracted from HRMECs were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes subsequently were blocked with 5% nonfat milk for 2 hours and incubated overnight with antibodies against caspase-1, IL-1β, NLRP1, NOX4, TXNIP, or NLRP3. After rinsing three times with TBST, the membranes were incubated with a secondary antibody at room temperature for 2 hours. β-Actin was used as an internal control. 
Flow Cytometry
Pyroptotic cell death was assessed using flow cytometry. Fluorochrome inhibitor of caspase-1 (caspase-1 FLICA) and propidium iodide (PI) were added to the HRMECs, and pyroptotic death was detected by flow cytometry after FLICA/PI staining. Pyroptosis is defined as PI (+) and caspase-1 FLICA (+). 
Caspase-1 Activity Assay
Cellular caspase-1 activity was assayed using a caspase-1 assay kit, according to the manufacturer's instructions. The results are presented as caspase-1 activity units per unit weight of protein. 
Immunofluorescence Staining
The cells were incubated with anti-caspase-1 and anti-IL-1β antibodies overnight and then incubated with a secondary antibody for 1 hour at room temperature in the dark. After rinsing three times with PBS, the slides were incubated with 4′,6-diamidino-2-phenylendole (DAPI) for 3 minutes and placed in glycerol. Fluorescence was detected using a fluorescence microscope. 
Luciferase Reporter Assay
A fragment of the NLRP1 or NOX4 3′-UTR containing the miR-590-3p binding sites was amplified by PCR and inserted at the XolI and NotI restriction sites, downstream of the luciferase coding sequence in the psi-CHECK2 vector (Promega). Cells seeded onto 12-well plates were transfected with the NLRP1 or NOX4 3′-UTR construct, together with the miR-590-3p mimic or its NC. After incubation for 24 hours, luciferase activity was detected using a Dual-Luciferase Reporter Assay System according to the manufacturer's instructions. 
Detection of Cellular ROS
HRMECs were seeded onto 24-well plates for 24 hours and then treated with the corresponding intervention. After treatment, the cells were rinsed and incubated with 5 μM DCFH-DA in the dark at 37°C for 30 minutes. Fluorescence was detected using a fluorescence microscope (488 nm excitation and 525 nm emission). 
Clinical Specimens
A total of 14 vitreous tissue samples, including five from DR patients, four from DM patients without DR, and five from patients without DM, were collected during pars plana vitrectomy at Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine from March 2019 to June 2019. Among the 14 patients, six had retinal detachment, five had epiretinal membranes, and three had a macular hole. This protocol was approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, and all patients were informed according to the World Medical Association Declaration of Helsinki. The demographic and clinical data of the study participants are shown in the Table
Table
 
Demographic and Clinical Characteristics of the Patients
Table
 
Demographic and Clinical Characteristics of the Patients
Enzyme-Linked Immunosorbent Assay (ELISA)
The level of IL-1β protein in vitreous tissues was determined using an IL-1β ELISA kit according to the manufacturer's instructions. 
Statistical Analysis
The data are presented as the mean ± SD. Differences among experimental groups were analyzed by 2-tailed Student's t-test or Fisher's exact test using GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) and SPSS (ver. 22.0; SPSS, Inc., Chicago, IL, USA). Notably, P < 0.05 was considered statistically significant. 
Results
HG Promoted Pyroptosis in HRMECs
HRMECs cultured under HG conditions were used to develop the DR cell model (Figs. 1A, 1B). Cell viability was significantly lower in the HG than in the control groups (Fig. 1C). To investigate the role of pyroptosis in DR, we detected changes in caspase-1 in HRMECs. As shown in Figures 1D and 1E, the pyroptosis rate in the HG group was markedly higher and caspase-1 activity was markedly increased. Moreover, Western blotting and immunofluorescence analyses showed that the expression of caspase-1 protein was markedly increased after HG treatment, indicating that HG promotes pyroptosis (Figs. 1F, 1G). 
Figure 1
 
HG conditions induce pyroptosis and the downregulation of miR-590-3p in HRMECs. (A) HRMECs observed under microscope. (B) Immunofluorescence analysis of factor VIII in HRMECs. (C) HRMECs were exposed to HG conditions and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (D) Cells were exposed to HG conditions for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (E) Caspase-1 activity was measured after the cells were exposed to HG conditions for 48 hours. (F) Western blot analysis of cleaved caspase-1 protein levels in cells of the HG and control groups. (G) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of HG and control groups. Data are presented as the mean ± SD. n = 3. **P < 0.01; ***P < 0.001.
Figure 1
 
HG conditions induce pyroptosis and the downregulation of miR-590-3p in HRMECs. (A) HRMECs observed under microscope. (B) Immunofluorescence analysis of factor VIII in HRMECs. (C) HRMECs were exposed to HG conditions and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (D) Cells were exposed to HG conditions for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (E) Caspase-1 activity was measured after the cells were exposed to HG conditions for 48 hours. (F) Western blot analysis of cleaved caspase-1 protein levels in cells of the HG and control groups. (G) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of HG and control groups. Data are presented as the mean ± SD. n = 3. **P < 0.01; ***P < 0.001.
miR-590-3p was Downregulated in Response to HG and Triggered Pyroptosis in HRMECs
We further investigated the potential involvement of miR-590-3p in HG-induced DR. qRT-PCR analysis revealed that miR-590-3p expression decreased after exposing HRMECs to HG (Fig. 2A). Furthermore, to identify the function of miR-590-3p in the response to HG, HRMECs transfected with the miR-590-3p mimic or inhibitor were exposed to HG conditions for 48 hours. Cell viability was significantly lower in the miR-590-3p inhibitor group (Fig. 2B) and pyroptosis was markedly higher (Figs. 2C–F). These phenomena were consistent with an HG-induced condition. Conversely, the miR-590-3p mimic rescued these effects of HG (Figs. 2G–K). These results demonstrated that miR-590-3p inhibited HG-induced pyroptosis in HRMECs. 
Figure 2
 
Downregulation of miR-590-3p in response to HG conditions triggers pyroptosis in HRMECs. (A) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to normal or HG conditions. (B) The viability of HRMECs transfected with the miR-590-3p inhibitor or inhibitor-NC for 24, 48, 72, and 96 hours was evaluated using a CCK8 assay. (C) Cells were transfected with inhibitor-NC or the miR-590-3p inhibitor for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after cells were treated with inhibitor-NC or the miR-590-3p inhibitor for 48 hours. (E) Western blot analysis of cleaved caspase-1 protein levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (G) HRMECs were transfected with a mimic-NC or the miR-590-3p mimic and then exposed to HG conditions for the viability assay. (H) Caspase-1 activity was measured in cells treated as indicated for 48 hours. (I) Western blot analysis of cleaved caspase-1 protein levels in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (J) Pyroptosis of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups were assessed using FLICA/PI staining and flow cytometric analysis. (K) Immunofluorescence analysis of caspase-1 and IL-β levels in cells treated as indicated. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
 
Downregulation of miR-590-3p in response to HG conditions triggers pyroptosis in HRMECs. (A) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to normal or HG conditions. (B) The viability of HRMECs transfected with the miR-590-3p inhibitor or inhibitor-NC for 24, 48, 72, and 96 hours was evaluated using a CCK8 assay. (C) Cells were transfected with inhibitor-NC or the miR-590-3p inhibitor for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after cells were treated with inhibitor-NC or the miR-590-3p inhibitor for 48 hours. (E) Western blot analysis of cleaved caspase-1 protein levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (G) HRMECs were transfected with a mimic-NC or the miR-590-3p mimic and then exposed to HG conditions for the viability assay. (H) Caspase-1 activity was measured in cells treated as indicated for 48 hours. (I) Western blot analysis of cleaved caspase-1 protein levels in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (J) Pyroptosis of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups were assessed using FLICA/PI staining and flow cytometric analysis. (K) Immunofluorescence analysis of caspase-1 and IL-β levels in cells treated as indicated. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Downregulation of miR-590-3p Promoted the NOX4 Pathway and NLRP1 in HRMECs
After further study, we found that HG treatment increased the expression of NOX4 and NLRP1 than in the control group, and the miR-590-3p mimic reversed these effects (Fig. 3A). Additionally, the expression levels of the downstream components of the NOX4 pathway—ROS, TXNIP, and NLRP3—were upregulated (Figs. 3A, 3B), suggesting a potential negative link between miR-590-3p and NOX4/NLRP1. We next performed a series of studies to investigate this relationship. Computational analysis by miRDB (available in the public domain at http://mirdb.org/) predicted a conserved binding site for miR-590-3p in the 3′-UTR of the NOX4 and NLRP1 genes (Figs. 3C, 3E). To verify that miR-590-3p directly targets NOX4 and NLRP1, we constructed luciferase reporter vectors carrying the NOX4 3′-UTR or the NLRP1 3′-UTR. We found that the miR-590-3p inhibitor upregulated the luciferase activity of the wild-type 3′-UTR compared to the mutant 3′-UTR (Figs. 3D, 3F). Conversely, the miR-590-3p mimic could downregulate the luciferase activity of the wild-type 3′-UTR compared to the mutant 3′-UTR (Figs. 3D, 3F). Thus, miR-590-3p targets the 3′-UTRs of NOX4 and NLRP1
Figure 3
 
miR-590-3p suppression in response to HG conditions promotes NLRP1 and the NOX4 pathway in HRMECs. (A) Western blot analysis of NLRP1 protein levels and the NOX4/TXNIP/NLRP3 pathway in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (B) Measurement of intracellular ROS generation in control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups using a DCFH-DA probe by fluorometry. (C) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NLRP1 was mutated to UUCCGG (MT). (D) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or the anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. (E) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NOX4 was mutated to UCCGGA (MT). (F) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. WT, wide type; MT, mutant. Data are the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
 
miR-590-3p suppression in response to HG conditions promotes NLRP1 and the NOX4 pathway in HRMECs. (A) Western blot analysis of NLRP1 protein levels and the NOX4/TXNIP/NLRP3 pathway in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (B) Measurement of intracellular ROS generation in control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups using a DCFH-DA probe by fluorometry. (C) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NLRP1 was mutated to UUCCGG (MT). (D) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or the anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. (E) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NOX4 was mutated to UCCGGA (MT). (F) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. WT, wide type; MT, mutant. Data are the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Overexpression of NOX4 Triggered Pyroptosis in HRMECs
Because miR-590-3p accelerated pyroptosis in HRMECs, directly inhibited NOX4, and increased the expression of downstream components of the NOX4 pathway, we were interested in exploring the pyroptosis-inducing function of NOX4. Based on qRT-PCR analysis, NOX4 mRNA levels were significantly higher in HRMECs transfected with NOX4 overexpression (OE-NOX4) plasmids (Fig. 4A). Cell viability was significantly lower in the presence of OE-NOX4 (Fig. 4B), and the pyroptosis rate was markedly higher (Fig. 4C). As shown in Figure 4D, caspase-1 activity markedly increased in the presence of OE-NOX4. Moreover, Western blotting and immunofluorescence analyses showed that caspase-1 protein expression was significantly higher (Figs. 4E, 4F), indicating that NOX4 overexpression induces pyroptosis. NOX4 is a ROS-generating enzyme, and the downstream components, including ROS, TXNIP, and NLRP3, were upregulated in the OE-NOX4 group (Figs. 4E, 4G), suggesting that NOX4 might induce pyroptosis via the ROS/TXNIP/NLRP3 axis. 
Figure 4
 
miR-590-3p modulates pyroptosis in HRMECs by targeting the NOX4 pathway and NLRP1. (A) qRT-PCR analysis of NOX4 levels in cells transfected with vector or OE-NOX4 plasmids. (B) HRMECs were transfected with vector or OE-NOX4 plasmids and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (C) Pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after the cells were transfected with plasmids as indicated for 48 hours. (E) Western blot analysis of TXNIP, NLRP3, and cleaved caspase-1 protein levels in cells of vector and OE-NOX4 groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of vector and OE-NOX4 groups. (G) Quantification of intracellular ROS generation in vector and OE-NOX4 cells. (H) The viability of HRMECs was evaluated using a CCK8 assay. siRNAs targeting NOX4 or NLRP1 were transfected into HRMECs before the miR-590-3p mimic treatment. HRMECs were transfected with oligos as indicated, exposed to HG and then tested for viability at 48 hours. (I) Quantification of pyroptotic death by flow cytometry after FLICA/PI staining. (J) Caspase-1 activity was measured after the cells were treated as indicated for 48 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 4
 
miR-590-3p modulates pyroptosis in HRMECs by targeting the NOX4 pathway and NLRP1. (A) qRT-PCR analysis of NOX4 levels in cells transfected with vector or OE-NOX4 plasmids. (B) HRMECs were transfected with vector or OE-NOX4 plasmids and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (C) Pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after the cells were transfected with plasmids as indicated for 48 hours. (E) Western blot analysis of TXNIP, NLRP3, and cleaved caspase-1 protein levels in cells of vector and OE-NOX4 groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of vector and OE-NOX4 groups. (G) Quantification of intracellular ROS generation in vector and OE-NOX4 cells. (H) The viability of HRMECs was evaluated using a CCK8 assay. siRNAs targeting NOX4 or NLRP1 were transfected into HRMECs before the miR-590-3p mimic treatment. HRMECs were transfected with oligos as indicated, exposed to HG and then tested for viability at 48 hours. (I) Quantification of pyroptotic death by flow cytometry after FLICA/PI staining. (J) Caspase-1 activity was measured after the cells were treated as indicated for 48 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
miR-590-3p Modulated Pyroptosis in HRMECs by Targeting NOX4 and NLRP1
We next used RNA interference to elucidate the mechanism by which miR-590-3p triggers pyroptosis. HRMECs were transfected with NOX4 or NLRP1 siRNA and exposed to HG. Cell viability was significantly increased in the si-NOX4 or si-NLRP1 group compared to the control group (Fig. 4H), and the pyroptosis rate was markedly decreased (Fig. 4I). Caspase-1 activity in the si-NOX4 or si-NLRP1 group also was markedly lower (Fig. 4J). These results indicated that NOX4 and NLRP1 siRNAs alleviate the pyroptosis induced by HG. When HRMECs were transfected with NOX4 and NLRP1 siRNAs simultaneously, adding the miR-590-3p mimic did not significantly improve cell activity and did not reduce the rate of pyroptosis, demonstrating that miR-590-3p modulates pyroptosis by targeting NOX4 and NLRP1
IL-1β Induced the Downregulation of miR-590-3p in HRMECs
Through analysis of clinical vitreous samples, we found that the expression of IL-1β was increased in DR patients, suggesting that pyroptosis has an important role in clinical DR (Fig. 5A). Moreover, the expression of IL-1β increased as the disease progressed, and therefore, we further explored the function of IL-1β on DR. Intriguingly, as the concentration of IL-1β increased, miR-590-3p expression significantly decreased (Fig. 5B) and the DNA promoter activity of pri-miR-590 also decreased, as detected using a luciferase reporter assay (Fig. 5C). This finding indicated that IL-1β downregulated miR-590-3p in DR by decreasing the DNA promoter activity of pri-miR-590
Figure 5
 
IL-1β induces the downregulation of miR-590-3p in HRMECs. (A) Pyroptosis in clinical DR specimens. Levels of IL-1β were measured in vitreous samples from nondiabetic control, DM patients, and DR patients using an ELISA kit. (B) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to IL-1β. (C) The DNA promoter activity of pri-miR-590 was measured after the cells were treated with IL-1β as indicated for 24 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
 
IL-1β induces the downregulation of miR-590-3p in HRMECs. (A) Pyroptosis in clinical DR specimens. Levels of IL-1β were measured in vitreous samples from nondiabetic control, DM patients, and DR patients using an ELISA kit. (B) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to IL-1β. (C) The DNA promoter activity of pri-miR-590 was measured after the cells were treated with IL-1β as indicated for 24 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Discussion
Inflammation has a crucial role in the progression of DR. Pyroptosis is a highly inflammatory form of programmed cell death characterized by cell swelling, pore formation, and nuclear condensation.57 Caspase-1 triggers pyroptotic death by cleaving the members of the gasdermin family, especially Gasdermin D, to induce pore formation, membrane rupture, and the release of IL-1β and IL-18.21 Thus far, this inflammatory-mediated process has been found in diabetic cardiomyopathy,22 diabetic nephropathy,23 and diabetic atherosclerosis.24 In our study, we found that the activity and expression of caspase-1 and IL-1β were significantly increased in HG-treated HRMECs and clinical vitreous samples, suggesting that pyroptosis is involved in the pathogenesis of DR. 
Increasing evidence has demonstrated that miRNAs function as effective biomarkers or therapeutic targets,25 and many miRNAs have been shown to have important roles in DR progression.26,27 In our study, we initially investigated the role in DR of miR-590-3p, which is an inflammation-related miRNA16,18,28 that may participate in the progression of pyroptosis. Our results indicated that the miR-590-3p inhibitor induced pyroptosis, which is consistent with the HG-induced condition. Moreover, the miR-590-3p mimic was able to rescue this alteration. These results confirmed that miR-590-3p is involved in pyroptosis in DR. 
Furthermore, we found that NOX4 and NLRP1 expression levels were increased in the HG group, and this effect could be partially corrected by treatment with the miR-590-3p mimic. Moreover, computational analysis predicted a conserved binding site for miR-590-3p in the 3′-UTR of the NOX4 and NLRP1 genes, indicating a potential negative relationship. In our study, we initially used luciferase reporter assays to confirm that miR-590-3p specifically targeted the NOX4 and NLRP1 3′-UTRs. 
NOX4 is a ROS-generating enzyme, and in a previous study, we showed that HG contributes to the increased vascular permeability and apoptosis in DR via the ROS/TXNIP/NLRP3 axis.11 NLRP1 and NLRP3 have important roles in pyroptosis.8 In our study, the expression levels of NLRP1, NOX4, TXNIP, and NLRP3 were upregulated in HG-treated HRMECs. The overexpression of NOX4 resulted in the upregulation of caspase-1 and IL-1β, which is consistent with an HG-induced condition. Furthermore, the production of ROS, TXNIP, and NLRP3 was increased after NOX4 overexpression. Moreover, when NOX4 or NLRP1 were silenced, cell viability was significantly increased, and the pyroptosis rate was markedly decreased. These results showed that NOX4 regulates NLRP3-dependent pyroptosis via the ROS/TXNIP axis and that NLRP1 promotes pyroptosis in DR. Chen et al.29 found that liraglutide attenuates NLRP3-dependent pyroptosis in cardiomyoblasts via the NOX4/ROS pathway, demonstrating a pyroptosis-inducing function of NOX4, which is consistent with our results. Additionally, when NOX4 and NLRP1 mRNA were simultaneously targeted by siRNAs, the addition of the miR-590-3p mimic did not significantly improve cell activity or reduce the pyroptosis rate, demonstrating that miR-590-3p modulates pyroptosis by targeting NOX4 and NLRP1
IL-1β, an important effector molecule in pyroptosis, is activated by increased levels of caspase-1.21 In our study, we initially showed that IL-1β upregulation downregulated miR-590-3p by decreasing the DNA promoter activity of pri-miR-590. This effect intensifies pyroptosis induced by decreased miR-590-3p levels through positive feedback, highlighting the significance of miR-590-3p in pyroptosis in DR. 
We initially confirmed that miR-590-3p was involved in pyroptosis of DR. Moreover, downregulated miR-590-3p promoted pyroptotic death by targeting NLRP1 and activating the NOX4-mediated pathway. Furthermore, we firstly showed that IL-1β upregulation downregulated miR-590-3p by decreasing the DNA promoter activity of pri-miR-590, which intensifies the pyroptosis through positive feedback (Fig. 6). However, this study still has several limitations. First, the mechanism whereby miR-590-3p regulates pyroptosis in vivo remains to be determined. Second, the number of clinical specimens included in this study was small. Nevertheless, this study expanded our knowledge of the function of miRNAs in pyroptosis and provided potential new biomarkers or therapeutic targets for DR. 
Figure 6
 
Diagrammatic sketch of the signaling pathway mediated by miR-590-3p to modulate pyroptosis in HRMECs.
Figure 6
 
Diagrammatic sketch of the signaling pathway mediated by miR-590-3p to modulate pyroptosis in HRMECs.
In conclusion, miR-590-3p levels are downregulated in DR, which promotes pyroptotic death. Moreover, increased levels of the pyroptosis-related cytokine IL-1β induce the further downregulation of miR-590-3p through positive feedback. Therefore, miR-590-3p might be an effective interfering target for the prevention and treatment of DR. 
Acknowledgments
Supported by the National Natural Science Foundation of China (81770947, 81970811), National Science and Technology Major Project of China (2017ZX09304010), and the Tibet Natural Science Foundation of China (XZ2017ZR-ZYZ09, XZ2018ZRG-95, XZ2018ZRG-100(Z)). 
Disclosure: C. Gu, None; D. Draga, None; C. Zhou, None; T. Su, None; C. Zou, None; Q. Gu, None; T. Lahm, None; Z. Zheng, None; Q. Qiu, None 
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Figure 1
 
HG conditions induce pyroptosis and the downregulation of miR-590-3p in HRMECs. (A) HRMECs observed under microscope. (B) Immunofluorescence analysis of factor VIII in HRMECs. (C) HRMECs were exposed to HG conditions and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (D) Cells were exposed to HG conditions for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (E) Caspase-1 activity was measured after the cells were exposed to HG conditions for 48 hours. (F) Western blot analysis of cleaved caspase-1 protein levels in cells of the HG and control groups. (G) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of HG and control groups. Data are presented as the mean ± SD. n = 3. **P < 0.01; ***P < 0.001.
Figure 1
 
HG conditions induce pyroptosis and the downregulation of miR-590-3p in HRMECs. (A) HRMECs observed under microscope. (B) Immunofluorescence analysis of factor VIII in HRMECs. (C) HRMECs were exposed to HG conditions and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (D) Cells were exposed to HG conditions for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (E) Caspase-1 activity was measured after the cells were exposed to HG conditions for 48 hours. (F) Western blot analysis of cleaved caspase-1 protein levels in cells of the HG and control groups. (G) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of HG and control groups. Data are presented as the mean ± SD. n = 3. **P < 0.01; ***P < 0.001.
Figure 2
 
Downregulation of miR-590-3p in response to HG conditions triggers pyroptosis in HRMECs. (A) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to normal or HG conditions. (B) The viability of HRMECs transfected with the miR-590-3p inhibitor or inhibitor-NC for 24, 48, 72, and 96 hours was evaluated using a CCK8 assay. (C) Cells were transfected with inhibitor-NC or the miR-590-3p inhibitor for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after cells were treated with inhibitor-NC or the miR-590-3p inhibitor for 48 hours. (E) Western blot analysis of cleaved caspase-1 protein levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (G) HRMECs were transfected with a mimic-NC or the miR-590-3p mimic and then exposed to HG conditions for the viability assay. (H) Caspase-1 activity was measured in cells treated as indicated for 48 hours. (I) Western blot analysis of cleaved caspase-1 protein levels in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (J) Pyroptosis of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups were assessed using FLICA/PI staining and flow cytometric analysis. (K) Immunofluorescence analysis of caspase-1 and IL-β levels in cells treated as indicated. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
 
Downregulation of miR-590-3p in response to HG conditions triggers pyroptosis in HRMECs. (A) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to normal or HG conditions. (B) The viability of HRMECs transfected with the miR-590-3p inhibitor or inhibitor-NC for 24, 48, 72, and 96 hours was evaluated using a CCK8 assay. (C) Cells were transfected with inhibitor-NC or the miR-590-3p inhibitor for 48 hours, and pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after cells were treated with inhibitor-NC or the miR-590-3p inhibitor for 48 hours. (E) Western blot analysis of cleaved caspase-1 protein levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of the inhibitor-NC and the miR-590-3p inhibitor groups. (G) HRMECs were transfected with a mimic-NC or the miR-590-3p mimic and then exposed to HG conditions for the viability assay. (H) Caspase-1 activity was measured in cells treated as indicated for 48 hours. (I) Western blot analysis of cleaved caspase-1 protein levels in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (J) Pyroptosis of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups were assessed using FLICA/PI staining and flow cytometric analysis. (K) Immunofluorescence analysis of caspase-1 and IL-β levels in cells treated as indicated. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
 
miR-590-3p suppression in response to HG conditions promotes NLRP1 and the NOX4 pathway in HRMECs. (A) Western blot analysis of NLRP1 protein levels and the NOX4/TXNIP/NLRP3 pathway in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (B) Measurement of intracellular ROS generation in control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups using a DCFH-DA probe by fluorometry. (C) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NLRP1 was mutated to UUCCGG (MT). (D) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or the anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. (E) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NOX4 was mutated to UCCGGA (MT). (F) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. WT, wide type; MT, mutant. Data are the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
 
miR-590-3p suppression in response to HG conditions promotes NLRP1 and the NOX4 pathway in HRMECs. (A) Western blot analysis of NLRP1 protein levels and the NOX4/TXNIP/NLRP3 pathway in cells of control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups. (B) Measurement of intracellular ROS generation in control, HG, HG+mimic-NC, and HG+miR-590-3p mimic groups using a DCFH-DA probe by fluorometry. (C) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NLRP1 was mutated to UUCCGG (MT). (D) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or the anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NLRP1-WT or NLRP1-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. (E) The potential miR-590-3p binding sites, UUUUAA (WT), in the 3′-UTR of NOX4 was mutated to UCCGGA (MT). (F) Left, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, inhibitor NC) or anti-miRNA of miR-590-3p (100 nM, miR-590-3p inhibitor) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Right, relative luciferase activity of the HRMECs transfected with NC oligos (100 nM, mimic-NC) or the miR-590-3p mimic (100 nM) plus the NOX4-WT or NOX4-MT luciferase reporter gene. Renilla luciferase activity served as an internal reference. WT, wide type; MT, mutant. Data are the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
 
miR-590-3p modulates pyroptosis in HRMECs by targeting the NOX4 pathway and NLRP1. (A) qRT-PCR analysis of NOX4 levels in cells transfected with vector or OE-NOX4 plasmids. (B) HRMECs were transfected with vector or OE-NOX4 plasmids and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (C) Pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after the cells were transfected with plasmids as indicated for 48 hours. (E) Western blot analysis of TXNIP, NLRP3, and cleaved caspase-1 protein levels in cells of vector and OE-NOX4 groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of vector and OE-NOX4 groups. (G) Quantification of intracellular ROS generation in vector and OE-NOX4 cells. (H) The viability of HRMECs was evaluated using a CCK8 assay. siRNAs targeting NOX4 or NLRP1 were transfected into HRMECs before the miR-590-3p mimic treatment. HRMECs were transfected with oligos as indicated, exposed to HG and then tested for viability at 48 hours. (I) Quantification of pyroptotic death by flow cytometry after FLICA/PI staining. (J) Caspase-1 activity was measured after the cells were treated as indicated for 48 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 4
 
miR-590-3p modulates pyroptosis in HRMECs by targeting the NOX4 pathway and NLRP1. (A) qRT-PCR analysis of NOX4 levels in cells transfected with vector or OE-NOX4 plasmids. (B) HRMECs were transfected with vector or OE-NOX4 plasmids and tested for viability at 24, 48, 72, and 96 hours using a CCK8 assay. (C) Pyroptosis was detected using FLICA/PI staining and flow cytometric analysis. (D) Caspase-1 activity was measured after the cells were transfected with plasmids as indicated for 48 hours. (E) Western blot analysis of TXNIP, NLRP3, and cleaved caspase-1 protein levels in cells of vector and OE-NOX4 groups. (F) Immunofluorescence analysis of caspase-1 and IL-β levels in cells of vector and OE-NOX4 groups. (G) Quantification of intracellular ROS generation in vector and OE-NOX4 cells. (H) The viability of HRMECs was evaluated using a CCK8 assay. siRNAs targeting NOX4 or NLRP1 were transfected into HRMECs before the miR-590-3p mimic treatment. HRMECs were transfected with oligos as indicated, exposed to HG and then tested for viability at 48 hours. (I) Quantification of pyroptotic death by flow cytometry after FLICA/PI staining. (J) Caspase-1 activity was measured after the cells were treated as indicated for 48 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 5
 
IL-1β induces the downregulation of miR-590-3p in HRMECs. (A) Pyroptosis in clinical DR specimens. Levels of IL-1β were measured in vitreous samples from nondiabetic control, DM patients, and DR patients using an ELISA kit. (B) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to IL-1β. (C) The DNA promoter activity of pri-miR-590 was measured after the cells were treated with IL-1β as indicated for 24 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5
 
IL-1β induces the downregulation of miR-590-3p in HRMECs. (A) Pyroptosis in clinical DR specimens. Levels of IL-1β were measured in vitreous samples from nondiabetic control, DM patients, and DR patients using an ELISA kit. (B) qRT-PCR analysis of miR-590-3p levels in HRMECs exposed to IL-1β. (C) The DNA promoter activity of pri-miR-590 was measured after the cells were treated with IL-1β as indicated for 24 hours. Data are presented as the mean ± SD. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
 
Diagrammatic sketch of the signaling pathway mediated by miR-590-3p to modulate pyroptosis in HRMECs.
Figure 6
 
Diagrammatic sketch of the signaling pathway mediated by miR-590-3p to modulate pyroptosis in HRMECs.
Table
 
Demographic and Clinical Characteristics of the Patients
Table
 
Demographic and Clinical Characteristics of the Patients
Supplement 1
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