Hsa_circ_0001879 promotes the progression of atherosclerosis by regulating the proliferation and migration of oxidation of low density lipoprotein (ox-LDL)-induced vascular endothelial cells via the miR-6873-5p-HDAC9 axis

ABSTRACT Atherosclerosis (AS) is a typical vascular disease. Emerging evidence has shown that circRNAs play key roles in the progression of AS, but the potential function and underlying mechanism of hsa_circ_0001879 remains unknown. We detected the expression level of hsa_circ_0001879 was determined by qRT-PCR, and the proliferation rate and migration ability of HUVECs were measured by CCK-8 assay and Transwell assay, respectively. Proliferative markers and epithelium mesenchymal transition (EMT) markers were measured through immunoblotting. A dual luciferase activity assay was performed to detect the interaction between circRNAs, miRNAs, and mRNAs. Hsa_circ_0001879 was upregulated in AS patients. Hsa_circ_0001879 inhibited the proliferation and migration ability of Human umbilical vein endothelial cells (HUVECs). Hsa_circ_0001879 directly bound to miR-6873-5p and acted as a sponge. miR-6873-5p-induced HDAC9 mRNA degradation was inhibited by hsa_circ_0001879. Hsa_circ_0001879 decreased the proliferation and migration of HUVECs by inhibiting miR-6873-5p-induced HDAC9 degradation.


Background
Atherosclerosis (AS) is a major chronic disease characterized by the formation of atherosclerotic plaques, which pose a high risk to the cardiovascular system [1]. During the progression of AS, endothelial cells (ECs) and smooth muscle cells are the key agents regulating the progression of AS [2][3][4]. The pathogenesis of AS can be summarized as follows: an increase in oxidation of low density lipoprotein (ox-LDL), the dysfunction of ECs and an increase in the apoptosis of ECs caused by mitochondrial damage [5]. Discovering the potential mechanism of ox-LDL in the progression of AS will help us better understand its pathogenesis.
circRNAs are noncoding RNAs formed through the back-splicing of pre-mRNAs. circRNAs do not have a polyA tail or cap [6]. With the advancement of high-throughput sequencing, circRNAs were reported to be involved in the progression of AS [7].
Hsa_circ_0001445 reverses ox-LDL-induced inhibition of HUVEC proliferation by targeting SRSF1 [8]. Inhibiting circ_UBR4 (hsa_circ_0010283) suppressed the proliferation, migration, and cell cycle progression of human vascular smooth muscle cells and promotes the progression of AS [9]. Circ_0004104 protects vascular endothelial cells from low-density lipoproteininduced dysfunction by targeting the miR-328-3p/ TRIM14 axis [10]. Circ_0093887 upregulates CCND2 and SUCNR1 expression to inhibit ox-LDLinduced endothelial cell dysfunction by functioning as a miR-876-3p sponge to compete with endogenous RNA [11], circ-SATB2 was upregulated in proliferating vascular smooth muscle cells (VSMCs) and regulated the phenotypic differentiation, proliferation, migration and apoptosis of VSMCs by miR-939-STIM1 axes [12], hsa_circ_0004104 was reported to regulated the macrophage function and was suggested as a potential therapy target for AS [13]. CicRNA circANRIL from the INK4/ARF locus was reported to regulate the expression of INK4/ARF and be associated with atherosclerotic vascular disease (ASVD) risk as well [14]. hsa_circ_0001879 was reported to be the biomarker of coronary artery disease [15], however, the potential function and underlying mechanism was still unknown.
In this study, we hypothesized that hsa_-circ_0001879 was engaged in the progression of AS and we tried to uncover the underlying mechanism, thus, we evaluated the expression of hsa_circ_0001879 in AS patients and found that hsa_circ_0001879 was upregulated. After establishing stable cell lines, the proliferation rate and migration ability of the cells were detected by CCK-8 assay and Transwell assay, respectively. Hsa_circ_0001879 inhibited the proliferation and migration of HUVECs. A dual luciferase activity assay showed that hsa_circ_0001879 inhibited miR-6873-5p-induced HDAC9 degradation by acting as a competing endogenous RNA.

Hsa_circ_0001879 was upregulated in AS patients and ox-LDL-treated HUVECs
We hypothesized that hsa_circ_0001879 was engaged in the progression of AS and we tried to uncover the underlying mechanism, thus, we evaluated the expression of hsa_circ_0001879 in AS patients and found that hsa_circ_0001879 was upregulated. After establishing stable cell lines, the proliferation rate and migration ability of the cells were detected by CCK-8 assay and Transwell assay, respectively. Hsa_circ_0001879 inhibited the proliferation and migration of HUVECs. A dual luciferase activity assay showed that hsa_circ_0001879 inhibited miR-6873-5p-induced HDAC9 degradation by acting as a competing endogenous RNA.

Hsa_circ_0001879 inhibited the proliferation and migration of ox-LDL-treated HUVECs
To determine the biology and underlying mechanism of hsa_circ_001879, we established stable overexpression and knockdown cell lines. The results are shown in Figure 2a (***, p < 0.001). We next detected the viability and migration ability of ox-LDL-treated HUVECs (100 mg/ml). The results showed that ox-LDL inhibited the proliferation (Figure 2b, p < 0.001) and migration of HUVECs (Figures 2c,Figures 2d, *, p < 0.05, **, p < 0.01, ***, p < 0.001). This inhibition was completely relieved by knocking down hsa_-circ_0001879. Overexpression of hsa_circ_0001879 had a cooperative effect with ox-LDL.

Hsa_circ_0001879 inhibited the expression of proliferation markers and the EMT
After showing that hsa_circ_0001879 inhibited the proliferation and migration of HUVECs, we measured the expression of PCNA and EMT markers, which are key markers reflecting the proliferation status and migration ability of cells. The results showed that PCNA expression was decreased in ox-LDL-treated cells and was restored in sh-hsa_circ_0001879 cells and decreased in hsa_circ_0001879-overexpressing cells ( Figure 3).

Hsa_circ_0001879 inhibited miR-6873-5pinduced HDAC9 degradation
By searching TargetScan, we identified HDAC9 as a potential target gene of miR-6873-5p. We first measured HDAC9 mRNA levels in cell lines. HDAC9 was decreased in hsa_-circ_0001879-knockdown cells at both the mRNA and protein levels (Figures 5a,Figures 5b, ***, p < 0.001). The potential binding site of HDAC9 and miR-6873-5p is shown in Figure 5c. We performed a dual luciferase activity reporting assay. The results showed that the luciferase activity increased in the MUT-HDAC9 cells compared with the control WT-HDAC9 cells, and this result further confirmed the interaction (Figure 5d, ***, p < 0.001). We next transfected a miR-6873-5p inhibitor into the hsa_circ_0001879-knockdown cell line and a miR-6873-5p mimic into hsa_circ_0001879- HUVECs were treated with ox-LDL with different concentration (mg/ml). The relative cell viability and apoptosis rate (c) was measured, *, p < 0.05, **, p < 0.01, ***, p < 0.001.D, HUVECs were treated with ox-LDL and the relative level of hsa_circ_0001879 was detected and normalized, *, p < 0.05, **, p < 0.01, ***, p < 0.001. overexpressing cells. The results showed that the expression of HDAC9 was completely restored in both cell lines (Figure 5d). We next measured HDAC9 expression in cells cotransfected with mut hsa_circ_0001879. HDAC9 remained unchanged in the cells transfected with mut hs_circ_0001879. The results indicated that hsa_-circ_0001879 inhibited miR-6873-5p-induced HDAC9 degradation (Figure 5e).

Patient information and clinical sample collection
AS samples and normal tissues were randomly collected from patients in the department of Cardiology, Guangdong Provincial Hospital of Traditional Chinese Medicine; The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. The including criteria was detailed as below: samples from patients with peripheral vascular AS were included and the samples were confirmed by pathologists. The normal tissues were normal peripheral vascular tissues from donors. This study was approved by the Ethics Committee of The Department of Cardiology, Guangdong Provincial Hospital of Traditional Chinese Medicine; The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. Clinical samples were collected from patients after written and informed consent was obtained.

Stable cell line construction
The hsa_circ_0001879 plasmid was generated by chemical synthesis of the complete sequence of hsa_circ_0001879, and additional circularization promoter ALU sequences were added upstream and downstream, the sequence was detailed as below: 5ʹTCTGACAACTGAACTGCTCTCGCCT TGAACCTGTTTTGGCACTAA AATAAAATCT GTTCAATTAACGAATTCTGAAATATGCTAT-CTTACAG -GTGAATATATTTTTTCTTGAGGA TCCACTAATTTGGGATGATAACGCCAAAAC-AGGTTCAAGGCGAGAGCAGTTCAGTTGTC-AGAA3ʹ, the sequence of hsa_circ_0001879 was cloned between AG and GT in the blank. The hsa_circ_0001879 shRNA-1 sequence was GGCTGTTCGGGAAAGTGTCAA, and the shRNA-2 sequence was CGGCTGTTCGGGAAA GTGTCA. The plasmids were transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's

Cell culture
Human umbilical vein endothelial cells (HUVECs) were acquired from the American Type Culture Collection. Cells were cultured with complete F12K (Sigma D0697) containing 10% fetal bovine serum (Invitrogen F8687). The cells were treated with different concentrations of ox-LDL for 48 h.

Dual-luciferase reporter assay
The fragment of hsa_circ_0001879 or HDAC9 3′ UTR containing the wild-type or mutant allele was inserted into the pmirGLO vector (LMAI Bio, Shanghai, China) to create a WT-hsa_circ_0001879, MUT-hsa_circ_0001879, WT-HDAC9, or MUT-HDAC9 reporter. The plasmids were transfected into HUVECs, and the relative luciferase activities were measured with a Dual-Lucy assay kit (Solarbio).

RNA Immunoprecipitation (RIP) assay
RIP analysis was performed using an EZ-Magna RIP kit (Millipore, Billerica, MA, USA) following the manufacturer's instructions. After lysing cells with RIP lysis buffer, cell lysates were incubated with magnetic beads coated with anti-Ago2 or anti-IgG (as the control). Additionally, qRT-PCR analysis was performed to measure the levels of hsa_circ_0001879 and miR-6873-5p.

Statistical analysis
All data analyses were performed with SPSS 20.0 statistical software. The differences between two groups were compared by Student's t-test. For multigroup analysis, one-way ANOVA followed by Tukey's post hoc test was performed when making comparisons within datasets. A p value <0.05 was considered to indicate a significant difference compared to the control.

Discussion
Evidence has demonstrated that endothelial cell dysfunction contributes to the progression of atherosclerosis [16,17]. The ox-LDL-treated HUVEC model has been well-established [18].
In our study, we identified a novel circRNA induced by ox-LDL and inhibited the proliferation and migration of HUEVCs through directly binds with miR-6873-5p and finally inhibited the degradation of HDAC9, EMT of endothelium cells and smooth muscle cells contributes critically to the progression of AS and understanding the underlying mechanism helped us better understand the disease [19].
CircRNAs play key roles in the progression of AS, especially in the function of endothelium vein cells. Hsa_circ_0030042 regulates autophagy and protects atherosclerotic plaque stability by targeting eIF4A3 [20]. The circular RNA circ_0003204 inhibits the proliferation, migration and tube formation of endothelial cells in atherosclerosis via the miR-370-3p/TGFβR2/ phosph-SMAD3 axis [21]. circGNAQ was reported to inhibit endothelial cell senescence and atherosclerosis progression [22].However, the potential mechanism of circRNA on endothelium cells was rarely studied.
In summary, circRNAs exert their functions in three main ways: as competing endogenous RNAs, RNA-binding protein partners and facilitators of peptide translation [23]. In the progression of AS, circRNAs mainly act as ceRNAs. Hence, we investigated the potential mechanism of hsa_circ_0001879 as a miRNA sponge. By investigating the circRNA database, we identified miR-6873-5p and HDAC9 as the potential target axis. Mutant hsa_circ_0001879 was established to confirm the interaction between hsa_-circ_0001879 and miR-6873-5p. We next detected miR-6873-5p-induced HDAC9 degradation. When we re-expressed a miR-6873-5p inhibitor and a miR-6873-5p mimic, the change in HDAC9 level was completely restored, indicating that hsa_circ_0001879 targets miR-6873-5p and thereby inhibits HDAC9 degradation.
The absence of HDAC9 attenuated AS progression by reducing inflammation and reversing cholesterol transport [24]. HDAC9 has been reported to be an independent biomarker useful for predicting the risk of AS [25]. HDAC9 activates IKK and thus regulates atherosclerotic plaque vulnerability [26]. HDAC9 has also been reported to regulate E2F3 and Rb1 and the progression of AS [27]. Most importantly, HDAC9 has been shown to be critical for ox-LDL-induced endothelial dysfunction [28]. Understanding the potential mechanism of HDAC9 in AS and its upstream regulation is urgently needed.
In conclusion, hsa_circ_0001879 increased the expression of HDAC9 by sponging miR-6873-5p, thereby preventing the cell growth and migration of HUVECs. These findings suggest that hsa_-circ_0001879 is a potential therapeutic target for AS.

Conclusion
Our study indicated that hsa_circ_0001879 promotes the progression of AS through inhibit miR-6873-5p induced HDAC degradation by acting as a competing endogenous RNA.

Notes on contributors
XW and QXC designed the article. All the authors were engaged in the experiment application, data analysis and manuscript writing and literal editing. All authors read and approved the final manuscript.