Circular RNA circ_0026218 Suppressed Atherosclerosis Progression via miR-338-3p/SIRT6 Axis

Background Multiple circular RNAs (circRNAs) are implicated in atherosclerosis (AS) pathogenesis. In fact, how circRNA 0026218 (circ_0026218) functions in AS remains unknown, and thus the functions and mechanisms of circ_0026218 in the injury of vascular endothelial cells are to be investigated. Methods Microarray analysis was employed to screen out differentially expressed circRNAs in AS. A cell model was mimicked by treating Human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (ox-LDL). circ_0026218, microRNA-338-3p (miR-338-3p) and silent information regulator 6 (SIRT6) expressions in HUVECs with ox-LDL treatment were probed by qRT-PCR. The cell proliferative capabilities were exposed by CCK-8 assay. The contents of interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) were measured by ELISA. Oxidative stress kits were utilized to detect the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). Flow cytometry was adopted to analyze the level of apoptosis of HUVECs. Dual-luciferase reporter gene assay and RIP assay were leveraged to expose the interplay between miR-338-3p and circ_0026218 or SIRT6 3′-UTR, respectively. In addition, the impacts of circ_0026216 and miR-338-3p on SIRT6 protein expressions were subjected to Western blot. Results circ_0026218 was greatly depleted in ox-LDL-stimulated HUVECs. circ_0026218 overexpression promoted viability of HUVECs in vitro and inhibited inflammatory response, oxidative stress, and apoptosis. circ_0026218 could adsorb miR-338-3p and positively modulated SIRT6 expressions via sponging miR-338-3p. Upregulation of this miRNA reversed the influence of circ_0026218 overexpression on ox-LDL-caused injury and apoptosis of HUVECs. Conclusion Collectively, circ_0026218 upregulates SIRT6 expression through decoying miR-338-3p, thereby inhibiting ox-LDL-initiated injury of HUVECs. circ_0026218 is involved in the pathogenesis of AS.


Introduction
Atherosclerosis (AS) appears as the pathological basis of diverse cardiovascular and cerebrovascular diseases such as cerebral infarction, coronary artery disorder, and peripheral vascular disease [1]. Endothelial cells have important functions in maintaining vascular homeostasis; their dysfunction is an early biological event of AS [2]. Various adverse stimuli (e.g., stress, inflammation, and hypoxia) can lead to endothelial cell injury and apoptosis in AS [3,4]. Oxidized low-density lipoprotein (ox-LDL) is a vital pathological part of AS. ox-LDL causes endothelial cell dysfunction and apoptosis, which triggers the formation of AS plaques [5]. Therefore, elucidation of ox-LDL-initiated endothelial cell dysfunction is pivotal for curing AS.
Circular RNA (circRNA) is a category of endogenous noncoding RNA transcripts with a covalently closed loop structure [6]. Reportedly, circRNAs feature prominently in multiple cardiovascular diseases [7]. For example, circ_0004543 is highly expressed in ox-LDL-initiated human umbilical vein endothelial cells (HUVECs), and circ_0004543 depletion increases the growth of ox-LDL-caused HUVECs [8]. circ_ 0065149 restrains ox-LDL-caused apoptosis and inflammation of HUVECs via targeting miR-330-5p [9]. circTM7SF3 promotes ox-LDL-mediated injury of macrophages by sponging miR-206 to raise ASPH expression [10]. In this work, circRNA_0026218 (circ_0026218) was abnormally depleted in phorbol 12-myristate 13-acetate-induced macrophages by analyzing circRNA microarrays (GSE107522). Macrophages' differentiation into foam cells is the key biological process in the pathogenesis of AS, and this result suggests that circ_ 0026216 participates in AS development. However, how circ_0026218 functions in the injury of endothelial cell is not clear.
MicroRNAs (miRNAs/miRs) are conceptually a category of small noncoding RNA transcripts of 21-25 nt that modulate gene expressions posttranscriptionally by restraining the translation of target mRNAs [11]. As reported, miRNAs feature prominently in the pathological processes of AS, lipoprotein metabolism, endothelial cell viability and apoptosis, and immune response included [12]. MicroRNA-338-3p (miR-338-3p) impairs the multiplication and interferes with the progression of gastric cancer [13], and glioblastoma [14]. Reportedly, in AS, miR-338-3p alleviates endothelial cell injury caused by ox-LDL via targeting BAMBI and initiating the TGF-β/Smad pathway [15]. Nonetheless, the potential mechanism that miR-338-3p mediates AS development has not been elucidated. Silent information regulator 6 (SIRT6), part of the sirtuin family, is a histone/nonhistone deacetylase that utilizes NAD + as a cofactor [16]. As reported, SIRT6 can prevent vascular smooth muscle cells from senescence and inhibit the development of AS [17]. Moreover, SIRT6 overexpression attenuates microcholesterol crystal-induced endothelial dysfunction [18]. Nevertheless, the mechanisms of SIRT6 dysregulation in endothelial cells need to be further elucidated.
This work is aimed at investigating the expression features and biological function of circ_0026218 in endothelial cells during the development of AS, and exploring its relations with miR-338-3p and SIRT6.

Materials and Methods
2.1. Bioinformatics Analysis. The microarray dataset GSE107522 was available from the Gene Expression Omnibus database, and microarray data analysis was accomplished as described previously [19]. Briefly, the interactive web tool GEO2R was leveraged to analyze the expression profile of cir-cRNAs in macrophages treated with 100 nM phorbol 12myristate 13-acetate, and stimulated with or without ox-LDL (50 mg/mL). circRNAs with P < 0:05 and log 2 ðfoldchangeÞ > 1 or < -1 were reflective of significant differences.
2.5. Ribonuclease (RNase) R Assay. 2 mg of total RNA samples were incubated for 15 min at 37°C in the presence or absence of 3 U/mg RNase R (Epicenter Biotechnologies, Madison, WI, USA). Next circ_0026218 and GAPDH expressions were detected by qRT-PCR.
2.6. Subcellular Distribution Assay. Nuclear and cytoplasmic RNAs were extracted from HUVECs using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). circ_0026218, U6, and GAPDH were then detected by qRT-PCR, with U6 and GAPDH as nuclear and cytoplasmic controls, respectively.    2.12. RNA Immunoprecipitation (RIP) Assay. RIP analysis was fulfilled by the Magna RIP RNA binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA). 100 μL of RIP lysis buffer, 0.5 μL protease inhibitor mixture and 0.25 μL RNase inhibitor were applied to prepare the complete RIP lysis buffer, which were mixed with HUVECs (5 × 10 5 cells) for 1 h. Then the lysates were followingly incubated overnight at 4°C with magnetic beads coupled with argo-naute2 (Ago2) antibody (Abcam) or immunoglobulin G (IgG) antibody (Abcam). The lysates were instantly treated with Proteinase K buffer, and the immunoprecipitated RNA was then extracted by the RNeasy MinElute purification kit (Qiagen, Shanghai, China), and circ_0026218 and miR-338-3p expressions in the immunoprecipitate were detected by qRT-PCR.
2.13. Western Blot Assay. HUVECs were initially lysed with RIPA lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride (PMSF; Beyotime) and total cellular proteins were harvested. Protein extracts were collected and quantified by the BCA protein assay kit (BioRad, Hercules, CA, USA).
2.14. Statistical Analysis. Data were in the form of mean ± standard deviation. These data were tackled with SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Discrepancies between two or among more groups were evaluated by Student's t-test or one-way ANOVA followed by Tukey's posthoc test. P < 0:05 is of statistical significance.

ox-LDL Inhibits circ_0026218 Expressions in HUVECs.
By tackling the microarray dataset GSE107522 in the public database, we found that circ_0026218 was downregulated in the in vitro model of ox-LDL-caused AS (Figure 1(a). To expound how circ_0026218 impacts the ox-LDL-initiated HUVECs, we probed into the impact of ox-LDL on circ_0026218 expressions in HUVECs under discrepant concentrations (0, 50, 75, and 100 μg/mL) or different treatment times (0, 24, 48, and 72 h) by qRT-PCR, demonstrating that circ_0026218 expressions were gradually reduced concentration-dependently (Figure 1(b) and time-dependently (Figure 1(c). To verify the circularity of circ_0026218, we performed RNase R assay, which displayed that RNase R decreased the level of GAPDH mRNA, but circ_0026218 level was not significantly impacted ( Figure 1(d). The analyses on the subcellular distribution of circ_0026218 displayed that circ_0026218 was mainly located in the cytoplasm of HUVECs (Figure 1(e), suggesting that circ_0026218 may interfere with the injury of endothelial cells and the development of AS.

Circ_0026218 Overexpression Enhances Viability, and Restrained Inflammatory Response, Oxidative Stress Damage and Apoptosis of HUVECs with ox-LDL Treatment.
To elaborate on the part of circ_0026218 in vascular endothelial cells, circ_0026218 overexpression vector (circ_ 0026218-oe) and control vector were meticulously transfected into HUVECs. We discovered that circ_0026218-oe transfection upregulated circ_0026218 expression in HUVECs (Figure 2 could play an inhibitory role in ox-LDL-caused injury of HUVECs.

SIRT6 Is a Downstream Target of miR-338-3p.
To elucidate the promising mechanism of circ_0026218/miR-338-3p axis in modulating ox-LDL-caused injury of vascular endothelial cells, we predicted the hidden targets of miR-338-3p by TargetScan database and discovered that this miRNA had a complementary binding site with SIRT6 (Figure 4(a). As dual-luciferase gene reporter assay confirmed, miR-338-3p overexpression significantly inhibited the activity of SIRT6-WT, while that of SIRT6-MUT was not significantly influenced (Figure 4(b). qRT-PCR and Western blot displayed that ox-LDL restrained SIRT6 mRNA and protein expressions in HUVECs concentration-and timedependently (Figures 4(c)-4(f)). In addition, miR-338-3p overexpression remarkably depressed SIRT6 mRNA and protein expressions in HUVECs as against those of the miRcon group (Figures 4(g) and 4(h); circ_0026218 overexpression increased SIRT6 mRNA and protein expressions in HUVECs as against the control group (Figures 4(g) and 4(h)).

Discussion
Vascular endothelial cell dysfunction is a pivotal hallmark of AS development. Recently, circRNAs were reported to regulate the phenotype of endothelial cells. For example, circ_ 0065149 overexpression restrains ox-LDL-induced injury of HUVECs and suppresses inflammatory responses and apoptosis [9]. circ_0001445 overexpression reverses the mitigated viability of HUVECs caused by ox-LDL via activation of the SRSF1/β-catenin axis [20]. circ_0001445 overexpression restrains ox-LDL-initiated functional impairment and apoptosis in HUVECs [21]. This work focused on how circ_0026218 functions in HUVECs with ox-LDL. We proved that ox-LDL restrained circ_0026218 expressions in HUVECs concentration-and time-dependently. In addition, circ_0026218 overexpression enhanced the viability of HUVECs and inhibited inflammatory response, oxidative stress and apoptosis, suggesting that circ_0026218 may have an ameliorative effect on vascular endothelial cells and thus inhibit AS progression.

BioMed Research International
MiRNAs are promising candidates for the diagnosis and therapy of cardiovascular diseases [22]. Some studies have also covered vital roles of miRNAs in modulating the injury of vascular endothelial cells. For instance, miR-92a-3p accelerates ox-LDL-induced apoptosis of HUVECs via modulating the SIRT6/MAPK signaling pathway [23]. MiR-126-5p strengthens endothelial viability and limits AS via depressing DLK1 [24]. Reportedly, circRNAs can sponge miRNA to regulate the biological behavior of cells [25]. Additionally, the circRNA-miRNA-mRNA axis has been widely reported to interfere with the pathogenesis of AS [26,27]. To elucidate the regulating mechanism of circ_0026218 in ox-LDL-stimulated injury of HUVECs, we predicted the target miRNAs of circ_0026218 by bioinformatics analysis and discovered that miR-338-3p could be decoyed by circ_0026218. The function of miR-338-3p has been covered in cancer biology in many previous studies. For instance, miR-338-3p is lowly expressed in prostate cancer [28], liver cancer [29], and colorectal cancer [30] and it inhibits tumor progression. MiR-338-3p is elevated in the aortic tissues of rat model with AS and it facilitates AS development [31]. This work proved that miR-338-3p was abundantly expressed in HUVECs treated with ox-LDL, which was coherent to the findings of the previous work [15]. Furthermore, miR-338-3p overexpression rescued the impacts of circ_0026218 upregulation on HUVECs, indicating that circ_0026218 could ameliorate ox-LDL-mediated injury of HUVECs via targeting miR-338-3p.
SIRT6 is a highly conserved NAD ( + )-dependent deacetylase involved in DNA damage repair, telomere stabilization, glucose and lipid metabolism, and oxidative stress [32]. Reportedly, SIRT6 features prominently in cardiovascular disease [33]. Specifically, SIRT6 enhances angiogenesis and hemorrhage of carotid plaque via modulating HIF-1α and reactive oxygen species [34]. SIRT6 reduces the generation of foam cells by prompting autophagy and cholesterol efflux under ox-LDL condition [35]. This study found that SIRT6 was lowly expressed in HUVECs with ox-LDL, which was coherent to the preceding study [23]. In addition, SIRT6 could be a downstream target of miR-338-3p in HUVECs. We found that circ_0026218 could upregulate SIRT6 expression in HUVECs by adsorbing miR-338-3p, showing that circ_0026218/miR-338-3p/SIRT6 regulatory network may be involved in AS progression.
But there are certain limitations. First, we only used the ox-LDL-induced injury model of HUVECs for our study. We did not assess whether circ_0026218 could modulate the injury of HUVECs induced by other factors; secondly, we only concentrated on the protective effect of circ_ 0026218 in vitro. Animal models should be considered in following studies to validate our findings; finally, other targets of circ_0026218 need to be expounded.
On all accounts, circ_0026218 expressions were inhibited in HUVECs treated with ox-LDL. Mechanistically, circ_0026218 upregulated SIRT6 via targeting miR-338-3p, thus promoting the viability and inhibiting the inflammatory response, oxidative stress, and apoptosis of HUVECs. This work reveals new potential molecular mechanisms in AS progression and provides an emerging theoretical basis for treating AS.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no competing interests.