Elsevier

Cytokine

Volume 136, December 2020, 155243
Cytokine

LncRNA-SNHG29 inhibits vascular smooth muscle cell calcification by downregulating miR-200b-3p to activate the α-Klotho/FGFR1/FGF23 axis

https://doi.org/10.1016/j.cyto.2020.155243Get rights and content

Highlights

  • SNHG29 decreases the expression of osteoblast-related factors to inhibit VC.

  • SNHG29 acts as a negative regulator of miR-200b-3p.

  • MiR-200b-3p inactivates α-Klotho/FGFR1/FGF23 axis in the process of VC.

  • SNHG29 downregulates miR-200b-3p to activate α-Klotho/FGFR1/FGF23 axis.

Abstract

Background

Vascular calcification (VC) is characterized by mineral accumulation on the walls of arteries and veins, which is a pathological process commonly found in elderly individuals and patients with atherosclerosis, hypertension, and diabetes. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play an important role in VC. However, the role of SNHG29 is less clear.

Methods

The expression of SNHG29, miR-200b-3p, α-Klotho, FGFR1 and FGF23 in vascular smooth muscle cells (VSMCs) was quantified by qRT-PCR and western blot assays. β-GP was used to construct an in vitro calcification model, followed by MTT assay to detect cell viability. Calcification was determined by alizarin red S staining and quantified by calcification assay. ALP activity was investigated by ALP staining. The interactions among SNHG29, miR-200b-3p and α-Klotho were verified by luciferase assay.

Results

In the in vitro calcification model, SNHG29 was downregulated, while miR-200b-3p was upregulated. SNHG29 overexpression and miR-200b-3p knockdown significantly suppressed osteoblast-related factors (RUNX2 and BMP2), accompanied by activation of the α-Klotho/FGFR1/FGF23 axis, further inhibiting the formation of calcified nodules. Moreover, miR-200b-3p overexpression and α-Klotho knockdown reversed the SNHG29 overexpression-induced inhibitory effects on calcified VSMCs.

Conclusion

Our study is the first to demonstrate that SNHG29 could inhibit VSMC calcification by downregulating miR-200b-3p to activate the α-Klotho/FGFR1/FGF23 axis, suggesting SNHG29 as a novel therapeutic target for VC-associated diseases.

Introduction

Vascular calcification (VC) refers to mineral accumulation on the walls of arteries and veins. Medial calcification is characterized by the deposition of hydroxyapatite in vascular smooth muscle cells (VSMCs), whose function is mainly to maintain a balance in vasoconstrictor tension. Thus, changes in the normal structure of VSMCs leads to medial calcification-associated vessel stiffness, systolic hypertension, and increased pulse wave velocity [1]. VC can be found in both the intimal and medial layers of the arteries throughout the human body, such as in heart valves, brain, kidney, bladder, and soft tissues. The occurrence of VC raises the risk of heart attack, stroke, dementia, renal insufficiency, etc [2], [3]. The prevalence of calcification increases with age, and it has been reported that over the age of 70 years, VC presents in 90% of men and 67% of women [4], [5].

VC is believed to be a passive process; however, recently, it has been demonstrated to be an active process involving the regulation of cellular signalling pathways, which is principally driven in part by VSMCs [1]. VSMCs have been shown to differentiate into osteoblast-like cells accompanied by the release of matrix vesicles to mediate calcium–phosphate deposition in blood vessels [6], [7]. In addition, osteoblast-related transcription factors, including msh homeobox2 (Msx2), SRY-box transcription factor 9 (Sox9) and Runt-related transcription factor 2 (RUNX2), have been shown to be highly upregulated in populations of VSMCs [6], [8]. In addition, mature VSMCs undergo phenotype switching from a contractile to a proliferative phenotype. The deregulation of phenotype switching is implicated in various vascular disorders, such as atherosclerosis [9]. Therefore, clarifying the key regulatory mechanism of VSMCs involved in VC is crucial to reduce vessel wall mineralization and accordingly alleviate the associated diseases.

Long non-coding RNAs (lncRNAs), defined as a family of transcribed RNA molecules over 200 nucleotides in length that are not translated into proteins, are correlated with biological development and the progression of various diseases [10], [11], [12]. LncRNA small nucleolar RNA host gene 29 (SNHG29) has been reported to be involved in osteosarcoma and colorectal carcinoma [13], [14]. However, the role of SNHG29 in VC is less understood. A previous study showed that SNHG29 is a negative regulator of VC and reduced VSMC calcification while decreasing the expression of osteoblast-related factors [8]. However, no information on the specific regulatory mechanism was provided.

The main accepted mechanism of lncRNAs is crosstalk with various proteins and microRNAs (miRNAs) [15]. Many miRNAs are involved in the regulation of VC. For instance, miRNA-221 and miRNA-222 are reported to synergistically promote VC [16]. The upregulated expression of miRNA-30b is correlated with the alleviation of VC [17]. MiR-200b-3p, which belongs to the miR-200 miRNA family, was reported to participate in multiple regulatory loops in different diseases, such as lung adenocarcinoma, pancreatic cancer, and prostate cancer [18], [19], [20]. However, the role of miR-200b-3p is poorly understood in VC-associated diseases. Recently, Kong demonstrated that miR-200b-3p was highly expressed in degenerative aortic stenosis [21] but did not provide specific regulatory mechanisms. We identified the putative binding sites between SNHG29 and miR-200b-3p based on the trusted databases starBase (http://starbase.sysu.edu.cn/index.php) and DIANA (http://www.microrna.gr/tarbase), which have been referenced in various impactful studies [22], [23]. Since the role of SNHG29 and miR-200b-3p in VSMCs has never been characterized, we performed a series of experiments to explore their respective functions during the VC process.

Alpha Klotho (α-Klotho), a 130-kDa single-pass transmembrane protein, was originally recognized as an anti-ageing protein [24]. In recent years, it has become clear that α-Klotho is a cofactor for fibroblast growth factor (FGF23) and FGF receptor (FGFR) that integrates a multistep regulatory system of minerals, including phosphorus and calcium [25], [26], [27]. Among various pathophysiological inducers of VC, phosphate is one of the most prominent [28]. The deficiency of α-Klotho triggers human artery calcification and mediates resistance to FGF23 [29]. In our previous study, we demonstrated that in VSMCs, the α-Klotho/FGF23 axis mediated high phosphate-induced VC. High phosphate-induced VC was significantly reversed by overexpression of α-Klotho and FGF23 [30]. Additionally, starBase also predicted α-Klotho as a potential binding target of miR-200b-3p. However, there are no reports that the α-Klotho/FGFR1/FGF23 axis is associated with miR-200b-3p.

In this study, we first characterized the SNHG29-mediated regulation of miR-200b-3p that inhibits the calcification of VSMCs through activation of the α-Klotho/FGF23/FGFR axis. Therefore, our study provides new insights into the molecular function of SNHG29 in the pathogenesis of VC and highlights the potential of lncRNAs to serve as new therapeutic targets in VC.

Section snippets

Cell culture

Primary human aortic smooth muscle cells (HASMCs) and human coronary artery smooth muscle cells (HCASMCs) were purchased from Thermo Fisher Scientific (Massachusetts, USA) for use in the study. Both cell lines were cultured in DMEM growth medium containing smooth muscle growth supplement (SMGS) (ScienCell, CA, USA) at 37 °C, 5% CO2, and 95% air in a humidified cell culture incubator.

In vitro calcification of VSMCs

VSMCs were cultured in DMEM growth medium. The calcification medium (DMEM growth medium containing 10 mM

SNHG29 and miR-200b-3p were differentially expressed in the β-GP-Induced in vitro vascular smooth muscle calcification model

We first treated primary HASMCs and HCASMCs with 10 mM β-GP to induce in vitro calcification. To elucidate the cytotoxic effect of β-GP, we measured cell viability and observed decreased viability depending on the exposure time in both cell lines (Fig. S1A). Calcification was confirmed by alizarin red S staining and calcification assays. The stained particles were diffusely scattered over the cell layer and increased over time (Fig. S1B&C). On the other hand, since VSMCs undergo a phenotypic

Discussion

VC, which is known to increase the risk of myocardial infarction and death, is a pathological process commonly found in elderly individuals and patients with hypertension, atherosclerosis, and diabetes [33]. Calcification can be found in the intimal and medial layers of arteries. The former is associated with blocked arteries or ruptured atherosclerotic plaque [34]. In contrast, the latter is associated with stiffed vessels, systolic hypertension or increased pulse wave velocity [1]. The

CRediT authorship contribution statement

Chong Huang: Conceptualization, Formal analysis, Methodology, Supervision, Validation, Writing - original draft. Jin-Feng Zhan: Formal analysis. Yan-Xia Chen: Investigation. Cheng-Yun Xu: Methodology, Supervision, Validation. Yan Chen: Data curation, Project administration, Writing - review & editing.

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.

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