Circ-SATB2 upregulates STIM1 expression and regulates vascular smooth muscle cell proliferation and differentiation through miR-939

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Highlights

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    Circ-SATB2 and STIM1 were up-regulated in proliferative VSMCs, while miR-939 were down-regulated.

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    Circ-SATB2 inhibits miR-939 transcriptional activity and upregulates its target gene STIM1 expression.

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    Circ-SATB2 regulates proliferation and differentiation of VSMCs through miR-939/STIM1.

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    Circ-SATB2 promotes migration of VSMCs through miR-939/STIM1.

Abstract

The prevention and treatment of coronary heart disease (CHD) is a difficult problem to be solved. More and more studies have found that circular RNAs (circRNAs) may play important roles in the development of CHD. Here detection of vascular smooth muscle cells (VSMCs) showed that circ-SATB2 and STIM1 were up-regulated in proliferative VSMCs, while miR-939 were down-regulated. Circ-SATB2 and miR-939 did not affect the expression of each other, but circ-SATB2 could promote while miR-939 inhibited the expression of STIM1 (a target gene of miR-939). Circ-SATB2 overexpression could inhibit the expression of SM22-alpha (SM22α, a marker of contractile VSMCs), while the expression of SM22α was promoted by miR-939. STIM1 could promote cell proliferation and migration, and circ-SATB2 had similar effects, but its linear sequence had no such functions. MiR-939 had the opposite effects, could promote cell apoptosis and inhibit cell proliferation and migration, and siRNAs targeting circ-SATB2 had similar effects. When co-transfected with circ-SATB2 over-expression vector and miR-939 mimics or STIM1 siRNAs, the changes of cell proliferation, apoptosis and migration were not significant. Therefore, circ-SATB2 can regulate VSMC phenotypic differentiation, proliferation, apoptosis and migration by promoting the expression of STIM1. This discovery will provide a theoretical reference for exploring the role of circRNA in VSMCs and the pathogenesis of CHD.

Introduction

Coronary atherosclerotic heart disease is one of the highest morbidity and mortality diseases in the world, which seriously threatens human health. According to the World Health Organization, by 2030, the number of coronary heart disease (CHD) deaths will increase to 23.3 million, will become the leading cause of human death [1]. Therefore, finding effective treatment for CHD is a difficult problem to be solved at present.

CHD is the most common type of organ disease caused by atherosclerosis (AS), and many atherosclerotic processes are closely related to the occurrence of CHD [2]. Vascular smooth muscle cells (VSMCs) are one of the main cells that constitute the structure of vascular wall and maintain vascular tension. Their structural and functional changes are the cytopathological basis of many cardiovascular diseases, such as hypertension, AS and restenosis after angioplasty [3]. Therefore, plaque formation, intimal thickening and vascular hypertrophy caused by phenotypic transformation of VSMCs are still the focus of current research [3]. The study on the mechanism of VSMCs phenotypic transformation has potential guiding value for reversing vascular remodeling and clinical prevention.

Circular RNA (circRNA) is a non-coding RNA (ncRNA) discovered in recent years, and a prospective research direction in cardiovascular field [4]. For example, Burd et al. [5] found that the expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. But so far, there are few reports on circRNAs and VSMCs. Therefore, we screened the circRNAs in VSMCs, and the expression of hsa_circ_0007422 (circ-SATB2, emanating from the SATB2 gene) was significantly up-regulated in proliferative VSMCs, which may be of great significance. More and more evidences show that circRNAs are involved in biological processes such as transcription, mRNA splicing, RNA degradation and translation [6], and are closely related to the occurrence and development of cancer, nervous system disorders, cardiovascular diseases and other human diseases [7]. Therefore, further study on the function and pathogenesis of these circRNAs may provide a new harrow point for the treatment of cardiovascular diseases.

CircRNAs have a variety of biological functions, and mainly serve as microRNA (miRNA) sponges, through interaction with miRNAs and thus associated with a variety of diseases [8]. Predictive analysis based on some biological software shows that miR-939 have multiple binding sites on circ-SATB2, and stromal interaction molecule 1 (STIM1) is an important target gene of miR-939. Although the study of miR-939 in VSMCs has not been reported, STIM1 is closely related to the proliferation of VSMCs [9,10]. Takahashi et al. [9] pointed out that down-regulation of STIM1 inhibited the proliferation of VSMCs cultured in vitro by blocking Ca2+ influx. Therefore, circ-SATB2/miR-939/STIM1 pathway may play an important role in the differentiation and proliferation of VSMCs, which is worthy of further study.

Section snippets

Cell culture

Human aortic smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured with high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) in a 37 °C humidified atmosphere containing 5% CO2. At approximately 70% confluency, cells were serum-starved in DMEM containing 0.1% FBS for 24 h. After that, cells were added with Platelet-derived growth factor

MiR-939 targeted binding to circ-SATB2 and STIM1, and their expression in VSMCs

CircRNA usually plays a regulatory role by acting as a sponge for miRNAs. Predictive analysis by some biological software (RegRNA 2.0 and RNAhybrid) indicates that miR-939 have multiple binding sites on circ-SATB2, and STIM1 is an important target gene of miR-939 (Fig. 1A). After cloning the linear sequence of circ-SATB2 into psiCHECK-2 vector, this plasmid and miR-939 mimics were co-transfected into cells. And then dual luciferase reporter gene assay showed that the luciferase activity was

Discussion

Differentiation and phenotypic transformation of VSMCs are critical initial steps in cardiovascular diseases such as hypertension, atherosclerosis and restenosis after angioplasty [11]. Therefore, it is important to study the molecular mechanism of VSMC differentiation and phenotypic regulation for the prevention and treatment of these diseases.

In this study, circRNAs expression in VSMCs was detected for the first time, and circ-SATB2 was upregulated in proliferative VSMCs and down-regulated in

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81273878).

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