Elsevier

Gene

Volume 747, 15 July 2020, 144653
Gene

Research paper
circRNA_0084043 contributes to the progression of diabetic retinopathy via sponging miR-140-3p and inducing TGFA gene expression in retinal pigment epithelial cells

https://doi.org/10.1016/j.gene.2020.144653Get rights and content

Highlights

  • circRNA_0084043 was significantly elevated in DR cell model.

  • Loss of circRNA_0084043 protected ARPE-19 cell against HG-induced cell damage.

  • miR-140-3p was identified as a direct target of circRNA_0084043.

  • TGFA was a target of miR-140-3p.

Abstract

Diabetic retinopathy (DR) is a frequent complication of diabetes and it can lead to visual impairment and blindness. However, the mechanism of their regulation remains little known. circRNAs can function as crucial competing endogenous RNA, which can sponge corresponding miRNAs and affect mRNA expression in various diseases, including DR. In our current research, we observed that circRNA_0084043 was elevated in high glucose (HG)-incubated ARPE-19 cells. Then, we focused on whether and how circRNA_0084043 participated in retinal vascular dysfunction under conditions diabetes. Apoptosis, inflammation and oxidative stress are hallmark of DR progression. This work was aimed to investigate the signaling mechanisms of circRNA_0084043 in these pathogenesis of DR. We discovered loss of circRNA_0084043 significantly increased cell survival and repressed HG-triggered apoptosis. In addition, knockdown of circRNA_0084043 remarkably reduced oxidative stress as evidenced by the down-regulated malondialdehyde (MDA) content, enhanced activities of Super Oxide Dismutase (SOD) and Glutathione peroxidase (GSH-PX). Addition, silence of circRNA_0084043 effectively restrained HG-stimulated inflammation as proved by repressing inflammatory cytokines Tumor Necrosis Factor α (TNF-α), Interleukin 6 (IL-6) and Cox-2 in ARPE-19 cells. Subsequently, we successfully predicted that miR-140-3p was a downstream target of circRNA_0084043, which could be negatively regulated by circRNA_0084043. Mechanistically, loss of miR-140-3p abrogated the beneficial effects of circRNA_0084043 siRNA on ARPE-19 cells. Transforming Growth Factor alpha (TGFA) can exhibit a role in multiple diseases. Taken these together, these data demonstrated that loss of circRNA_0084043 depressed HG-induced damage via sponging miR-140-3p and regulating TGFA.

Introduction

Diabetes mellitus is a chronic disease with great morbidity and mortality across the global (Santos et al., 2017). Diabetic retinopathy (DR) is a serious microvascular complication of diabetes all over the world (Murchison et al., 2017). As well established, many metabolic disorders are closely associated with the pathogenesis of DR (Arroba et al., 2018). Currently, the molecular mechanisms of DR progression are still poorly known and no effective treatments have been developed for DR since now. Our current research was aimed to identify risk factors for DR and indicate a novel target.

Circular RNA is abundantly expressed in eukaryotes and can exert important biological roles (Kristensen et al., 2018, Rybak-Wolf et al., 2015). Increasing evidence has shown that circRNA can function as a ceRNA via sponging miRNAs (Hansen et al., 2013, Zhong et al., 2018). Clinical data has displayed that circRNAs are involved in a variety of diseases, which suggest that circRNAs has a wide effect on diseases (Zhou et al., 2018, Zhuo et al., 2020, Li et al., 2020). For example, circ-CMPK1 can contribute to lung cancer cell proliferation through sponging miR-302e (Cui et al., 2019). CircRNA ITCH can repress the progression of prostate cancer through inducing HOXB13 and sponging miR-17-5p (Wang et al., 2019). Recently, the clinical significance of circRNAs has been shown in DR (Zhang et al., 2017, Gu et al., 2017). For instance, loss of circRNA DMNT3B results in diabetic retinal vascular dysfunction via regulating miR-20b-5p and BAMBI (Zhu et al., 2019). However, the biological effect of circRNA_0084043 on DR progression remains barley known.

Currently, we demonstrated that circRNA_0084043 promoted cell proliferation and inhibited cell apoptosis induced by HG. Meanwhile, oxidative stress and inflammation were also repressed by loss of circRNA_0084043. Then, we further investigated the interaction between circRNA_0084043 and miR-140-3p, since miR-140-3p was predicted as a target of circRNA_0084043. Moreover, inhibitors of miR-140-3p successfully reversed the function of circRNA_0084043 siRNA on DR progression via targeting TGFA.

Section snippets

Cell culture

ARPE-19 was obtained from ATCC (Manassas, VA, USA). Cells were incubated in DMEM with 10% FBS (Sigma, St. Louis, MO, USA), 1% non-essential amino acids, 100 g/ml streptomycin (Invitrogen, Carlsbad, CA, USA) and 100U/ml penicillin (Invitrogen, Carlsbad, CA, USA) in 5% CO2 at 37 °C.

Cell transfection

miR-140-3p mimics (10 nM), inhibitors (100 nM), circRNA_0084043 (50 nM) or the corresponding negative controls were obtained from GenePharma (Shanghai, China). Transfection was carried out by Lipofectamine 3000

circRNA_0084043 was significantly elevated in DR cell model

Firstly, we established cell injury model of DR. circRNA_0084043 was tested in ARPE-19 cells treated with HG. The data as shown in Fig. 1A denoted that circRNA_0084043 was remarkably increased by HG in vitro.

Down-regulated circRNA_0084043 protected ARPE-19 cell against HG-inhibited cell proliferation and HG-induced cell apoptosis

Moreover, in order to study the role of circRNA_0084043 on HG-induced cell injury, ARPE-19 cells were infected with circRNA_0084043 siRNA. As shown in Fig. 2A, circRNA_0084043 was obviously increased by circRNA_0084043 siRNA in ARPE-19 cells. Results in Fig. 2B exhibited that cell viability

Discussion

Recently, circRNAs have obtained increasing attention due to their roles in gene modulation of various human diseases (Barrett and Salzman, 2016). Diabetic vascular complications remain the leading causes of blindness (Tabit et al., 2010, Sena et al., 2013). Meanwhile, diabetes-triggered retinal vascular dysfunction is a main cause of blindness (Stitt et al., 2016). In our study, we observed that circRNA_0084043 was elevated in ARPE-19 cells incubated with HG. In addition, we found that loss of

Data Availability Statement

The data are available upon reasonable request.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Ying Li: Writing - original draft, Methodology, Data curation. Ting Chen: Software, Methodology, Supervision, Validation, Visualization. Chengliang Wan: Investigation, Writing - original draft, Data curation, Formal analysis, Software, Methodology, Supervision, Validation, Visualization. Hongyan Cang: Conceptualization, Project administration, Writing - review & editing, Resources.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

None.

References (34)

  • L.L. Santos et al.

    Use of SGLT-2 inhibitors in the treatment of type 2 diabetes mellitus

    Rev. Assoc. Med. Bras.

    (2017)
  • Murchison AP, Hark L, Pizzi LT, Dai Y, Mayro EL, Storey PP, et al. Non-adherence to eye care in people with diabetes....
  • A.I. Arroba et al.

    IGF-1, inflammation and retinal degeneration: a close network

    Front. Aging Neurosci.

    (2018)
  • L.S. Kristensen et al.

    Circular RNAs in cancer: opportunities and challenges in the field

    Oncogene

    (2018)
  • T.B. Hansen et al.

    Natural RNA circles function as efficient microRNA sponges

    Nature

    (2013)
  • Y. Zhong et al.

    Circular RNAs function as ceRNAs to regulate and control human cancer progression

    Molecular cancer.

    (2018)
  • C.J. Zhuo et al.

    Circular RNAs in early brain development and their influence and clinical significance in neuropsychiatric disorders

    Neural Regener. Res.

    (2020)
  • Cited by (50)

    • CircRNA SCMH1 regulates the miR-200a-3p/ZEB1 signaling axis to promote diabetes-induced retinal epithelial-mesenchymal transition

      2022, Experimental Eye Research
      Citation Excerpt :

      aberrant levels of circRNAs were observed in DR patients compare to non-diabetes patients (He et al., 2020). In addition, circRNA_0084043 regulated DR progression in RPE cells (Li et al., 2020). Thus, exploring the mechanisms of circRNAs in DR progression will facilitate developing new therapies.

    • CircFAT1 regulates retinal pigment epithelial cell pyroptosis and autophagy via mediating m6A reader protein YTHDF2 expression in diabetic retinopathy

      2022, Experimental Eye Research
      Citation Excerpt :

      In recent years, more and more attention has been paid to the role of circRNAs in the occurrence and development of DR. Knockdown of hsa_circ_0002570 inhibited HG–induced angiogenesis and inflammation in retinal microvascular endothelial cells (Liu et al., 2020). Inhibition of circRNA_0084043 promoted the progression of DR via regulating miR-140-3p and TGFA (Li et al., 2020). In this study, through circRNA sequencing, we identified 189 DEcircRNAs in retinal proliferative fibrovascular membranes of DR patients.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text