Circ_0002984 induces proliferation, migration and inflammation response of VSMCs induced by ox‐LDL through miR‑326‐3p/VAMP3 axis in atherosclerosis

Abstract Atherosclerosis can result in multiple cardiovascular diseases. Circular RNAs (CircRNAs) have been reported as significant non‐coding RNAs in atherosclerosis progression. Dysfunction of vascular smooth muscle cells (VSMCs) is involved in atherosclerosis. However, up to now, the effect of circ_0002984 in atherosclerosis is still unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low‐density lipoprotein (ox‐LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up‐regulated in the serum of atherosclerosis patients and ox‐LDL‐incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNF‐α and IL‐6 levels. The data implied that lack of circ_0002984 obviously repressed ox‐LDL–stimulated VSMC inflammation. Meanwhile, miR‐326‐3p, which was predicted as a target of circ_0002984, was obviously down‐regulated in VSMCs treated by ox‐LDL. Additionally, after overexpression circ_0002984 in VSMCs, a decrease in miR‐326‐3p was observed. Subsequently, miR‐326‐3p was demonstrated to target vesicle‐associated membrane protein 3 (VAMP3). Therefore, we hypothesized that circ_0002984 could modulate expression of VAMP3 through sponging miR‐326‐3p. Furthermore, we confirmed that up‐regulation of miR‐326‐3p rescued the circ_0002984 overexpressing‐mediated effects on VMSC viability, migration and inflammation. Additionally, miR‐326‐3p inhibitor‐mediated functions on VSMCs were reversed by knockdown of VAMP3. In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR‐326‐3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.

unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low-density lipoprotein (ox-LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNFα and IL-6 levels. The data implied that lack of circ_0002984 obviously repressed ox-LDL-stimulated VSMC inflammation.
In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR-326-3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.

| INTRODUC TI ON
Atherosclerosis is a common cause of various vascular diseases. 1 The disease resulted from atherosclerosis is becoming a serious concern. Atherosclerosis can induce formation of thrombus through disrupting the integrity of arterial surface. 2 It is significant to identify the possible mechanism of atherosclerosis development. Its common lesions of atherosclerosis can include sclerosis, stenosis and atherosclerotic plaque formation. 3,4 Recently, VSMCs are reported to participate in the remodelling of arterial wall. 5 The viability and migration of VSMCs act a crucial role in atherosclerosis progression. 6,7 Therefore, it is significant to find out the detailed mechanisms of atherosclerosis.
CircRNAs contain a covalently closed continuous loop, and they are single-stranded RNAs. 8 CircRNAs are resistant to the degradation mediated by exonuclease. 9 Many studies report that there is a close link between atherosclerosis progression and cir-cRNAs. 10,11 For instance, circ-Sirt1 can control NF-κB through enhancing SIRT1 via binding to miR-132/212 in VSMCs. 12 In addition, circ_0003204 can repress endothelial cell proliferation and migration through sponging miR-370-3p in atherosclerosis progression. 13 Previously, microarray analysis has indicated that circ_0002984 is elevated in VSMCs treated with ox-LDL. 14 The mechanism of circ_0002984 in atherosclerosis progression needs more investigation.

MicroRNAs can represent a kind of endogenous non-coding
RNAs with approximately 19-22 nucleotides. 15 Through complementarily binding their seed sequences with their targeting mRNA, mi-croRNAs transcriptionally inhibit it to modulate post-transcriptional gene expression. 16 MicroRNAs can exhibit crucial roles in regulating atherosclerosis. 17,18 Recently, it has been reported that the loss of miR-326-3p participates in various diseases. However, whether miR-326-3p may exhibit a regulatory role in VSMCs remains poorly known.
Based on these backgrounds, our research aimed to explore the biological function of circ_0002984 in atherosclerosis. We hypothesize that circ_0002984 regulates VAMP3 expression mainly through miR-326-3p to promote atherosclerosis progression. In our study, we implied that circ_0002984 was increased in atherosclerosis, which resulted in the down-regulation of miR-326-3p in VSMCs, consequently leading to the enhancement of VAMP3 and the progression of AS.

| Clinical samples
Our study was done based on the approval of Medical Ethical  Table 1.
Serums were extracted through 1000 g centrifugation at room temperature. Informed consents from participants were obtained before enrolment.
Cell viability was tested using a CCK-8 assay (Beyotime). In brief,

| Cell cycle analysis
Cells were fixed using 1 mL cold 70% ethanol for a whole night at −20°C before measuring. Then, the centrifugation of suspension was carried out at 1000 g for 5 minutes and we removed the ethanol. Cells were washed using PBS, and afterwards, PI solution in the presence of 1% RNase A was added. After incubated with no light at 37°C, cell cycle analysis was tested using flow cytometry.

| Transwell assay
Transwell chambers (Beijing Solarbio) were employed to assess cell migratory capacity. Cells were grown on the upper chamber.
Then, the medium with 10% FBS was loaded in the lower chamber. After crystal violet was added for 10 minutes, the cells were counted using an inverted light microscope (Olympus). Five visual fields were randomly selected to calculate the number of cells. All experiments were carried out 3 times, and the results were exhibited using average values.

| qRT-PCR
Total RNA from clinical samples and cells was extracted by TRIzol reagent and reverse-transcribed to cDNA using a PrimeScript RT Master Mix kit (Takara Biotechnology). The qPCR was performed using a SYBR ® Green PCR Master Mix (Vazyme Biotech). PCR was carried out in a reaction volume of 10 µL, with 5 µL 2× PCR master mix (SYBR Premix Ex Taq), 0.5 µL of PCR Primer, 2 µL of cDNA and diluted to 20 µL ddH 2 O. The quantitative real-time reaction was set at an initial denaturation step of 3 minutes at 94℃; and 94℃ 10s, 58℃ 40s, 94℃ 10s in 45 cycles, with a step from 58 to 94℃. Data were analysed using 2 − ΔΔC t method. Meanwhile, circ_0002984, VAMP3, IL-6 and TNFα expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). U6 was utilized as the control of miR-326-3p.
Primers were displayed in Table S1.

| Western blot
Proteins were isolated by radio immunoprecipitation (RIPA) buffer.

Protein concentration was tested using Bradford Protein Quantification
Kit (Vazyme Biotech). Then, equal protein was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membrane.
Afterwards, the membranes were blocked with 5% skimmed milk for 1 hour. The membranes were incubated with the primary antibodies: anti-VAMP3 and GAPDH (Abcam) at 4°C. Next day, the membranes were incubated using secondary antibody for 2 hours. Subsequently, the membranes were analysed using an electrochemiluminescence (ECL) kit (Vazyme Biotech) on ChemiDoc™ MP Imaging System.

F I G U R E 1
Circ_0002984 expression in VSMCs exposed to ox-LDL. A, Relative expression of circ_0002984 in the serum of atherosclerosis patients (n = 30) and healthy controls (n = 30). B, VSMCs were incubated with 20-100 μg/mL of ox-LDL. C, VSMCs were treated with ox-LDL for 0, 6, 12, 24, 36 or 48 h. D, Expression of circ_0002984 secreted in culture medium in the VSMC incubated with 100 mg/L ox-LDL for 24 h. Compared with the control group, *P < 0.05; **compared with healthy group, *P < 0.001

| Enzyme-linked immunosorbent (ELISA) assay
The supernatants of VSMCs were collected, and concentrations of cytokines (TNFα and IL-6) were evaluated by ELISA kits (R&D Systems).

| Statistical analysis
Data were analysed by GraphPad Prism 7. Student's t test was utilized to analyse the differences between two independent groups. Then, oneway analysis of variance was carried out to assess the differences among multiple groups. Tukey's post hoc test was carried out following ANOVA P < 0.05 was considered to demonstrate statistical significance.

| Circ_0002984 expression was elevated in VSMCs induced by ox-LDL
Firstly, we detected circ_0002984 expression in atherosclerosis patients. As shown in Figure 1A, circ_0002984 was significantly increased in atherosclerosis serum (P < 0.001). To study whether circ_0002984 exhibited a role in VSMCs, we assessed whether circ_0002984 level was modulated by ox-LDL. Firstly, VSMCs were incubated with ox-LDL at various concentrations (0, 20, 40, 60, 80 or 100 mg/L) to mimic atherosclerosis in vitro. We observed that circ_0002984 was increased by ox-LDL exposure ( Figure 1B, P < 0.05). Then, 100 mg/L was used to treat VSMCs and circ_0002984 was tested at different time points. A time-dependent enhancement of circ_0002984 was displayed in response to ox-LDL ( Figure 1C, P < 0.05). In Figure 1D, we have shown an up-regulation of circ_0002984 secreted in culture medium in the VSMC incubated with 100 mg/L ox-LDL for 24 hours (P < 0.05). In conclusion, circ_0002984 was increased in atherosclerosis, and herein, it was important to investigate the effect of circ_0002984.

| Circ_0002984 increases the proliferation and migration of VSMCs
Next, the function of circ_0002984 in ox-LDL-loaded VSMCs was focused on. We demonstrated that circ_0002984 was more F I G U R E 3 Circ_0002984 induces IL-6 and TNFα production following ox-LDL stimulation. A, Correlation between mRNA expression of IL-6 and circ_0002984 in human patients. B, Correlation between mRNA expression of TNFα and circ_0002984 in human patients. C, D, IL-6 and TNFα mRNA levels were determined by RT-qPCR. E, F, Concentration of IL-6 and TNFα in VSMCs was tested using ELISA assay. Compared with the control group, *P < 0.05 resistant to RNase R in comparison with linear UBR4 mRNA ( Figure 2A, P < 0.05). In Figure 2B, circ_0002984 expression was significantly reduced by circ_0002984 siRNA in VSMCs (P < 0.05).

| Circ_0002984 activates IL-6 and TNFα releases following ox-LDL treatment
Inflammatory process plays a key role in atherosclerosis. We observed a positive correlation between IL-6 and circ_0002984 as shown in Figure 3A (P < 0.05). A positive correlation between TNFα and circ_0002984 was also indicated in Figure 3B. Then, RT-qPCR and ELISA were used to evaluate the pro-inflammatory cytokines including IL-6 and TNFα in VSMCs. In Figure 3C,D (P < 0.05), IL-6 and TNFα mRNA levels were reduced by loss of circ_0002984. In addition, in Figure 3E,F, concentration of IL-6 and TNFα in VSMCs increased by ox-LDL was significantly reversed after circ_0002984 siRNA transfection (P < 0.05).

| Circ_0002984 acted as a sponge of miR-326-3p in ox-LDL-induced VSMCs
To further understand the regulatory relationship between and miRNAs, the miRNA targets for circ_0002984 were predicted in our study by starBase database. In Figure 4A, miR-326-3p was predicted to have the binding sites of circ_0002984. In Figure 4B, miR-326-3p was increased by miR-326-3p mimics while decreased by miR-326-3p inhibitors (P < 0.05). Dual-luciferase and RIP assays were carried out. It was revealed miR-326-3p significantly reduced the luciferase activity of circ_0002984 WT ( Figure 4C, P < 0.05).

| D ISCUSS I ON
It has been revealed that atherosclerosis is a serious burden to public health. Meanwhile, some reports have found that dysfunction of VSMCs is involved in atherosclerosis. 19 be proved in vivo. In addition, the mechanism of up-regulation of circ_0002984 induced by ox-LDL in VSMCs is unclear and needs more investigation.
To sum up, circ_0002984 could regulate the cellular development of ox-LDL-incubated VSMCs. We validated the function of circ_0002984 on atherosclerosis via regulating miR-326-3p and VAMP3. Thus, circ_0002984 might serve as a potential target for atherosclerosis treatment.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.