Repression of microRNA-21 inhibits retinal vascular endothelial cell growth and angiogenesis via PTEN dependent-PI3K/Akt/VEGF signaling pathway in diabetic retinopathy

https://doi.org/10.1016/j.exer.2019.107886Get rights and content

Highlights

  • Roles of miR-21 in EC viability and angiogenesis of DR were studied.

  • MiR-21 targeted PTEN and repressed its expression.

  • Increased miR-21 and reduced PTEN expression were observed in rats with DR.

  • Increased angiogenesis was observed in retina of rats with DR.

  • Activated PI3K/Akt/VEGF axis induced VEGF mRNA transcription and expression.

Abstract

Diabetic retinopathy (DR) is a microvascular complication of diabetes and one of the most common causes of blindness in active stage. This study is performed to explore the effects of microRNA-21 (miR-21) on retinal vascular endothelial cell (RVEC) viability and angiogenesis in rats with DR via the phosphatidylinositiol 3-kinase/protein kinase B (PI3K/Akt)/vascular endothelial growth factor (VEGF) signaling pathway by binding to phosphatase and tensin homolog (PTEN). Sprague Dawley (SD) rats were used for establishment of DR models. Target relationship between miR-21 and PTEN was assessed by bioinformatics prediction in combination with dual-luciferase reporter gene assay. Identification of expression of miR-21, PTEN and PI3K/Akt/VEGF signaling pathway-related genes in the retinal tissues was then conducted. In order to assess the contributory role of miR-21 in DR, the RVECs were transfected with mimic or inhibitor of miR-21, or siRNA-PTEN, followed by the detection of expression of PTEN and PI3K/Akt/VEGF-related genes, as well as the measurement of cell viability, cell cycle and apoptosis. Increased expression of miR-21 and PI3K/Akt/VEGF related genes, along with a reduced expression of PTEN was observed in the retinal tissues of DR rats. PTEN was targeted and negatively regulated by miR-21, while the PI3K/Akt/VEGF signaling pathway was activated by miR-21. RVECs transfected with miR-21 inhibitor exhibited promoted viability and angiogenesis, and inhibited apoptosis. To conclude, our results indicated that miR-21 overexpression could potentially stimulate RVEC viability and angiogenesis in rats with DR through activation of the PI3K/Akt/VEGF signaling pathway via repressing PTEN expression, highlighting the potential of miR-21 as a target for DR treatment.

Introduction

Diabetic retinopathy (DR) arises in approximately one third of the diabetic population and is considered to be the main cause of blindness in adults aged between 24 and 70 years (Olivares et al., 2017). The incidence of DR increases along the course of the disease, influencing more than 60% of type 2 DM patients with DR history for more than 20 years (Capitao and Soares, 2016). In addition, the prevalence of DR has been approximately recorded between 15.3% and 42.4% where the incidence of DR is influenced by changeable risk factors including blood pressure, blood glucose, serum lipids and smoking, and unchangeable risk factors like age, duration, genetic predisposition, and ethnicity (Zhao et al., 2014). Current treatments for DR primarily reduce the risk of visual loss and blindness, such as tight glycemic and blood pressure control might decrease risk of vision loss and DR (Arevalo, 2013). Endothelial cells (ECs) are the main participants of retinal ischemic vasculopathies and their dysfunction consequently initiates DR microvascular disorder, and chronic inflammatory responses in the ECs of the retina lead to the production of inflammatory mediators, increased vascular permeability, endothelial cell apoptosis and angiogenesis (Wang et al., 2015). A previous study confirmed the interaction between miR-126 and insulin receptor substrate-1 (IRS-1), which presented that the results of this interaction in the retinal pericytes express a large number of insulin signal transduction-related proteins, including IRS-1 and phosphoinositide 3-kinase (PI3K) (Fang et al., 2017). Conventionally, the typical insulin signal transduction pathway comprises of a combination of IRS-1/PI3K/protein kinase B (Akt) and its downstream signaling molecules, which induces mRNA transcription of vascular endothelial growth factor (VEGF) and the expression of VEGF, thereby stimulating the formation of retinal neovascularization (Lu et al., 1999).

MicroRNA-21 (MiR-21) located on chromosome 17q23-2 has vital effects on cell viability, angiogenesis and anti-apoptosis, and also shares association with many physiological and pathophysiological processes such as angiogenesis, glucose homoeostasis, and the pathogenesis of diabetes and related microvascular and macrovascular complications (Jiang et al., 2017). Additionally, a previous study reported that miR-21 overexpression is responsible for the loss of peroxisome proliferator-activated receptor-α in DR (Chen et al., 2017). As a tumor-suppressor gene located at the chromosomal locus 10q23, phosphatase and tensin homolog (PTEN) functions in various cellular processes and its activity has been found to be irresponsive or mutated in series of cancers (Shinde and Maddika, 2016). The suppression of miR-21 increases the radio-sensitivity of esophageal cancer TE-1 cells, which is resulted from the activation of PTEN in esophageal squamous cell carcinoma (Huang et al., 2013). PTEN gene encodes for a protein phosphatase, which can negatively modulate PI3K/Akt signaling pathway, thus regulating cell development in a variety of healthy or tumorous tissues (Lin et al., 2016). VEGF is a pro-angiogenesis factor that can be mediated by the PI3K/Akt signaling pathway (Di et al., 2017). Angiogenesis, the formation of new blood vessels from pre-existing ones, is implicated in embryonic vascular development and may develop peripheral retinal vessels in the inner retina and outer plexus capillaries in the vessels formed in the central retinal vessels by vasculogenesis (Al-Shabrawey et al., 2013). A previous investigation documented elevation of the PI3K/Akt signaling pathway induced by retina-specific PTEN deficiency can lead to pathological changes of retinal interneurons (Sakagami et al., 2012). Although the important role of miR-21/PTEN axis has been demonstrated in other types of diabetic diseases, the underlying mechanism in DR still remains unclear. Hence, we hypothesized that suppression of miR-21 could re-express PTEN and inactivate the PI3K/Akt/VEGF pathway, ultimately preventing the progression of DR.

Section snippets

Ethics statements

This study was conducted in strict accordance with the recommendations stated in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiment protocol was approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Dalian Medical University.

Establishment of DR rat models and culture of retinal vascular endothelial cells (RVECs)

A total of 65 healthy adult male Sprague Dawley (SD) rats (weighing 190–235 g; aged 8 weeks) from the Shanghai Experimental Animal Center of Chinese Academy of Sciences

Lowly expressed PTEN and highly expressed PI3K, Akt, VEGF, and CD34 are observed in retinal tissues of DR rats

Initially, Immunohistochemistry was employed to measure the expression of PTEN, VEGF, and CD34 as well as the extent of PI3K and Akt phosphorylation. The results depicted in Fig. 1A and B showed that compared with the control group, the expression rate of PTEN in the DR group was lower, (all p < 0.05). Phosphorylated PI3K and Akt were mainly expressed in cell membrane and partly in cytoplasm, with a higher positive expression rate in the DR group than in the control group (p < 0.05). In

Discussion

DR, as a manifestation of diabetes is the main cause of blindness in the elderly worldwide, with increased incidence in Asian countries like China (Yau et al., 2012). RVEC dysfunction has been crucial in the etiology and pathogenesis of DR (Monaghan et al., 2015). Moreover, a study highlighted the correlation of miR-21 with the pathogenic process of type 2 diabetes with DR (Jiang et al., 2017). An increased miR-21 expression has been reported in the vitreous humor of proliferative DR, which

Declaration of competing interest

None.

Acknowledgments

This study was supported by Foundation of Liaoning Educational Committee (Grant No. LQ2017016), National Natural Science Foundation of China (Grant No. 81300737) and Natural Science Foundation of Liaoning Province of China (Grant No. 20170540274; Grant No. 20180550524). All authors thanked reviewers for their kind comments.

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