Circular RNA HIPK3 regulates human lens epithelial cells proliferation and apoptosis by targeting the miR-193a/CRYAA axis

https://doi.org/10.1016/j.bbrc.2018.06.149Get rights and content

Highlights

  • CircRNA sequencing reveals 26 up-regulated and 75 down-regulated circRNAs in ARC.

  • CircHIPK3 is down-regulated in all three subtypes of ARC.

  • CircHIPK3 knockdown affects the cellular function in primary cultured HLECs.

  • CircHIPK3 exerts its role via modulation of circHIPK3/miR-193a/CRYAA axis in HLECs.

Abstract

Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back splicing. Accumulating evidence has demonstrated their vital regulation in several biological processes and ocular diseases. However, the role of circRNAs in age-related cataract (ARC), the leading cause of visual impairment worldwide, is still unknown. CircRNA sequencing reveals that 101 circRNAs are differentially expressed between the capsules of transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs transcripts. Eight of 10 differentially expressed circRNAs are further verified by quantitative RT-PCRs. One highly conserved circRNA, circHIPK3, is significantly down-regulated in all cortical, nuclear and posterior subcapsular subtypes of ARC. The silencing of circHIPK3, but not HIPK3 mRNA, significantly accelerates apoptosis development upon oxidative stress and decreases cell viability and proliferation in primary cultured human lens epithelial cells (HLECs). The expression of α-SMA and vimentin was downregulated, while the expression of E-cadherin and ZO-1was upregulated, suggesting the repression of epithelial-mesenchymal transition after circHIPK3 knockdown. CircHIPK3 silencing increases miR-193a expression. miR-193a regulates CRYAA expression by targeting the binding site within the 3′UTR. Moreover, miR-193a decreases the viability and proliferation, and increases the apoptosis of HLECs upon oxidative stress. This study suggests that circRNAs are the potential regulators in cataractogenesis. CircHIPK3 regulates HLECs function through miR-193a-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of ARC.

Introduction

Age related cataract (ARC) is the leading cause of visual impairment and blindness on a global scale [1]. Surgical intervention is the most effective treatment option. However, with the accelerating trend of population aging, the rapid increase of demand for cataract surgery pose great economic burden for society [2]. Thus, it is still required to investigate the potential mechanisms in cataractogenesis for alternative pharmacological means to prevent or delay ARC development.

Circular RNAs (circRNAs) are a novel class of non-coding RNAs characterized by the covalently closed loop structure linking the 3′ and 5′ ends [3]. Circular RNAs participate in a vast range of developmental and physiological processes, including fibrosis, cell apoptosis, proliferation, differentiation, and angiogenesis [4]. Importantly, increasing evidence has shown that aberrant expression of circRNAs are closely associated with the pathogenesis of ocular diseases [[5], [6], [7], [8], [9]]. However, the characterization and function of circRNAs in ARC remain largely unknown.

In this study, we employed circular RNA sequencing technique to identify ARC-related circRNAs. We showed that 101 circRNAs were differentially expressed between transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs. Eight of 10 circRNAs were verified by qRT-PCR. Of them, we characterized one highly conserved circRNA, circHIPK3, and revealed that circHIPK3 knockdown could affect the viability, apoptosis and proliferation of human lens epithelial cells (HLECs), suggesting a potential role of circHIPK3 in ARC formation.

Section snippets

Ethic statement

The use of human lens samples from postmortem eyes and cataract eyes during surgery was approved by the institutional review board of Eye and ENT Hospital of Fudan University. This study was performed in accordance with the tenets of the Declaration of Helsinki for research involving human subjects. Written informed consents were obtained from every enrolled participant.

Human lens epithelium samples

Lens epithelium samples were obtained from the postmortem eyes (donor age range 50–61 years, free of ocular diseases,

Differential circRNA expression between transparent and ARC lens capsules

Circular RNA sequencing was used to identify ARC-related circRNAs. We set the threshold as the fold change >2.0, and identified 101 differentially expressed circRNAs, including 75 down-regulated circRNAs and 26 up-regulated circRNAs (ARC versus controls; Supplementary Table 2). We then conducted hierarchical clustering analysis to obtain a systematic comparison of circRNA expression between ARC and control group using the top 30 down-regulated and top 20 up-regulated circRNAs (Fig. 1A). Eight

Discussion

Circular RNAs have recently gained attention due to their key roles in the regulation of many biological processes and ocular disorders [4]. However, their roles in ARC are still unclear. In this study, we identified 101 differentially expressed circRNAs between ARC and transparent lens capsules. Of them, circRNA HIPK3 level was down-regulated in ARC capsules. In vitro study revealed the important role of circHIPK3/miR-193a/CRYAA network in regulating HLECs function.

ARC is characterized by

Conflicts of interest

The authors report no conflicts of interest in this work.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81371002).

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    Xin Liu, Baihui Liu and Menglong Zhou contributed equally to this work and should be considered as co-first authors.

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