circ_0003204 Regulates Cell Growth, Oxidative Stress, and Inflammation in ox-LDL-Induced Vascular Endothelial Cells via Regulating miR-942-5p/HDAC9 Axis

Background: Atherosclerosis (AS) is a typical inflammatory vascular disease. Many reports corroborated that circular RNAs (circRNAs) is involved in AS progression. However, the potential function and possible mechanism of circ_0003204 in AS progression remain indistinct. Methods: Expression level analysis was performed using qRT-PCR and western blot. Cell viability and apoptosis were determined using Cell Counting Kit-8 (CCK-8), flow cytometry, and western blot assays. The status of oxidative stress and inflammation was determined via commercial detection kits and ELISA assay, respectively. The binding relationship was verified via dual-luciferase reporter and RNA immunoprecipitation assays. Results: ox-LDL increased circ_0003204 and HDAC9 levels and decreased miR-942-5p level. Silencing of circ_0003204 enhanced cell viability and inhibited cell apoptosis, oxidative stress and inflammation in ox-LDL-disposed HUVECs. In addition, circ_0003204 targeted miR-942-5p to regulate ox-LDL-resulted HUVECs injury. Also, miR-942-5p affected ox-LDL-triggered HUVECs injury by targeting HDAC9. Furthermore, circ_0003204 elevated HDAC9 expression via decoying miR-942-5p. Conclusion: circ_0003204 aggravated ox-LDL-induced HUVECs damage via modulating miR-942-5p/HDAC9 pathway.

Circular RNAs (circRNAs) are neotype RNA molecules without 5 ′ to 3 ′ polarity produced by back-splicing (5). Mounting studies have corroborated that circRNAs are closely related to cardiovascular diseases (6), including AS. For example, down-regulation of circ_0029589 suppressed the growth and motility of ox-LDL-mediated VSMCs in AS via competitively combining with miR-424-5p to regulate IGF2 (7). Qin et al. (8) suggested that depletion of circ_0003645 mitigated ox-LDL-mediated apoptosis and inflammation in HUVECs via regulating NF-κB pathway. In addition, circ_0003204 was prominently increased in ox-LDL-disposed human aorta endothelial cells (HAECs), and up-regulation of circ_0003204 hindered HAECs growth and migration in AS (9). Also, Liu et al. revealed that circ_0003204 silencing accelerated ox-LDL-triggered proliferation and angiogenesis in HUVECs (10). Nonetheless, the exact function and mechanism of circ_0003204 in endothelial injury have not been elucidated.
Substantial literatures have demonstrated that circRNAs participate in the development of cardiovascular diseases via serving as microRNA (miRNA) sponges (11). Additionally, miRNAs contribute to target gene silencing and degradation via base-pairing with mRNA 3 ′ UTR (12,13). Moreover, increasing reports have verified that aberrantly expressed miRNAs exert crucial regulatory effects on many biological processes of AS (14,15). For instance, miR-151 restrained the apoptosis of endothelial cells in ox-LDL-induced HAECs via repressing Interleukin-17A (16). Qin et al. (17) found that miR-328-3p ameliorated ox-LDLresulted HUVECs injury in AS by binding to FOXO4. Besides, we predicted that circ_0003204 might target miR-942-5p through bioinformatics analysis. Also, Hua et al. (18) unveiled that miR-942 down-regulation overturned the inhibition of ZEB1-AS1 silencing on ox-LDL-induced endothelial injury.
Herein, we established an ox-LDL-disposed HUVECs model and investigated the expression pattern and biological function of circ_0003204 in ox-LDL-stimulated HUVECs. Furthermore, we explored the interaction between circ_0003204 and miR-942-5p/histone deacetylase 9 (HDAC9) pathway in ox-LDLmediated HUVECs.

Quantitative Real-Time PCR (qRT-PCR)
TRIzol reagent (Leagene, Beijing, China) was applied for extracting total RNA. Afterwards, cDNA was synthesized using the specific reverse transcription kit (Vazyme, Nanjing, China). For detecting RNA levels, qRT-PCR reactions were carried out using SYBR Green Master Mix (Vazyme). RNA levels were quantified via the 2 − Ct method. GAPDH (for circ_0003204 and HDAC9) and U6 (for miR-942-5p) were regarded endogenous controls. The primers included: circ_0003204-F: Cell Viability Assay 2 × 10 3 HUVECs were added into 96-well plates and then exposed to ox-LDL for the indicated time. Afterwards, the cells were incubated with 10 µL CCK-8 reagent (Boster, Wuhan, China) for 4 h. Finally, cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Flow Cytometry
Cell apoptosis was evaluated by adopting AnnexinV-FITC/Propidium Iodide (PI) Apoptosis Detection kit (Abcam, Cambridge, UK). HUVECs in 6-well plates were resuspended in binding buffer and then stained with AnnexinV-FITC and PI. Finally, the apoptosis analysis was conducted using flow cytometer (Beckman Coulter, Miami, FL, USA).

Measurement of MDA, SOD, and ROS
HUVECs treated with different conditions were harvested and lysed, and the supernatant was collected by centrifugation at 12,000 g for 5 min. Afterwards, malondialdehyde (MDA) level, superoxide dismutase (SOD) activity and reactive oxygen species (ROS) formation were determined using the corresponding kits (Abcam) according to the manufacturer's requirements.

Enzyme Linked Immunosorbent Assay (ELISA)
Transfected HUVECs were stimulated with ox-LDL for 48 h, and the culture medium were collected. Subsequently, the levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in HUVECs culture medium were examined using the specific ELISA kits (Boster) following the manufacturer's instructions.

RNA Immunoprecipitation (RIP) Assay
RIP analysis was implemented using EZ-Magna RIP kit (Millipore, Billerica, MA, USA). After lysing cells with RIP lysis buffer, cell lysates were incubated with magnetic beads combined with anti-Ago2 or anti-IgG (as the control). Additionally, qRT-PCR analysis was utilized to measure the abundance of circ_0003204 and miR-942-5p.

Statistical Analysis
Data were expressed as mean ± standard deviation (SD) in three independent replicates by using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Student's t-test or one-way analysis of variance was utilized to evaluate the differences between groups. P < 0.05 was considered statistically significant.

RESULTS circ_0003204 Expression Is Increased by ox-LDL in HUVECs
To explore the effect of ox-LDL on HUVECs, the cytotoxicity and apoptosis rate were tested in HUVECs exposed to different concentrations of ox-LDL (0, 25, 50, 75, and 100 mg/L). CCK-8 and flow cytometry assays showed that ox-LDL strikingly reduced the viability of HUVECs and increased the apoptosis rate of HUVECs in a dose-dependent manner (Figures 1A,B). Furthermore, we also examined the effect of ox-LDL at different doses on circ_0003204 expression in HUVECs. As depicted in Figure 1C, ox-LDL significantly increased the expression of circ_0003204 in a concentration-dependent manner. Therefore, the concentration of 100 mg/L was selected for subsequent experiments.

Knockdown of circ_0003204 Enhances Cell Viability and Inhibits Cell Apoptosis, Oxidative Stress and Inflammation in ox-LDL-Disposed HUVECs
First of all, qRT-PCR analysis showed that transfection of circ_0003204 siRNA remarkably reduced circ_0003204 expression, but had no effect on linear USP36 (Figures 2A,B). In addition, si-circ_0003204#1 with the most significant knockdown efficiency was selected for subsequent experiments. To investigate the role of circ_0003204 in ox-LDL-mediated damage, HUVECs transduced with si-NC or si-circ_0003204#1 were stimulated with 100 mg/ml ox-LDL. CCK-8 analysis showed that circ_0003204 silencing and ox-LDL stimulation strikingly increased the viability of HUVECs compared with ox-LDL treatment alone ( Figure 2C). In addition, ox-LDL-mediated down-regulation of PCNA expression  The levels of inflammatory cytokines were detected via ELISA. Data were presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001.
was restored by circ_0003204 depletion (Figure 2D and Supplementary Figure 3A). Flow cytometry suggested that down-regulation of circ_0003204 significantly decelerated ox-LDL-triggered apoptosis in HUVECs ( Figure 2E). In addition, the Bcl-2 family composed of pro-apoptotic (such as Bax) and anti-apoptotic (such as Bcl-2) members plays an important role in cell death (19). As expected, circ_0003204 knockdown markedly decreased Bax expression and increased Bcl-2 expression in ox-LDL-treated HUVECs (Figure 2F, Supplementary Figures 2A, 3B,C). Moreover, ox-LDL increased MDA level and ROS formation and decreased SOD activity, while these changes were reversed by suppressing circ_0003204 (Figures 2G-I). Besides, ELISA revealed that circ_0003204 silence attenuated the increase of IL-1β, IL-6, and TNF-α caused by ox-LDL stimulation in HUVECs (Figures 2J-L). Overall, these data indicated that silencing of circ_0003204 elevated cell viability and hindered cell apoptosis, oxidative stress and inflammation in ox-LDL-stimulated HUVECs.
Frontiers in Cardiovascular Medicine | www.frontiersin.org the expression levels of 6 candidate genes after miR-942-5p knockdown. The results showed that the up-regulation of HDAC9 was the most significant, so we chose HDAC9 as a possible target of miR-942-5p for follow-up studies (Supplementary Figure 1). As displayed in Figure 5A, miR-942-5p and HDAC9 3 ′ UTR possessed a putative binding site. Additionally, dual-luciferase reporter analysis suggested that miR-942-5p mimic significantly declined the luciferase activity of WT-HDAC9-3 ′ UTR reporter ( Figure 5B). Besides, ox-LDL remarkably increased the protein and mRNA expression of HDAC9 relative to the control group (Figure 5C and Supplementary Figure 4A). Western blot and qRT-PCR analysis exhibited that HDAC9 protein and mRNA levels in the pc-HDAC9 group were markedly elevated compared with the pc-NC group (Figure 5D and Supplementary Figure 4B). Furthermore, co-transfection with miR-942-5p mimic and pc-HDAC9 abrogated the reduction in HDAC9 protein and mRNA levels caused by miR-942-5p overexpression ( Figure 5E and Supplementary Figure 4C). These results evidenced that miR-942-5p directly targeted and negatively regulated HDAC9.

DISCUSSION
Mounting evidence has corroborated that endothelial dysfunction induces oxidative stress and inflammation, thereby promoting the development of AS (20,21). In addition, ox-LDL contributes to endothelial cell dysfunction and damage, as well as VSMCs growth and migration (22,23). Therefore, AS cell model can be established by stimulating HUVECs with ox-LDL. Besides, substantial investigations have demonstrated that circRNAs are recognized as critical mediators in AS progression (24). In addition, the cAMP, AMPK, and FOXO signaling pathways have been verified to be involved in the progression of atherosclerosis (25)(26)(27). In the current research, we unveiled that the new regulatory axis of circ_0003204/miR-942-5p/HDAC9 might provide a promising therapeutic target for AS.
Herein, we validated that circ_0003204 was conspicuously elevated in ox-LDL-treated HUVECs. Furthermore, circ_0003204 expedited HUVECs injury by inhibiting cell proliferation and inducing oxidative stress and inflammation under ox-LDL stimulation. In terms of mechanism, plentiful studies have manifested that circRNAs regulate various biological functions in AS via participating in competing endogenous RNA (ceRNA) network (28). For instance, circ_0010283 facilitated the FIGURE 7 | circ_0003204 regulates HDAC9 expression via sponging miR-942-5p. HUVECs were introduced with si-circ_0003204#1 or/and miR-942-5p inhibitor, and HDAC9 expression was tested using western blot. Data were presented as mean ± SD, n = 3. *P < 0.05, and **P < 0.01. growth and migration of ox-LDL-stimulated VMSCs through sponging miR-370-3p and elevating HMGB1 expression (29). Circ_0124644 aggravated endothelial injury triggered by ox-LDL stimulation in HUVECs via absorbing miR-149-5p to activate PAPP-A (30). Circ_CHFR facilitated ox-LDL-resulted cell growth, migration and inflammation in VSMCs by decoying miR-214-3p (31). Additionally, Zheng et al. (9) discovered that circ_0003204 restrained endothelial cell proliferation, migration and angiogenesis by sequestering miR-370-3p to regulate TGFβR2/phosph-SMAD3 signaling. However, the underlying mechanism of circ_0003204 in AS needs further investigation, and the purpose of this study is to discover a new circRNA-miRNA-mRNA axis involved in the pathogenesis of AS.
Hence, we investigated the potential ceRNA mechanism of circ_0003204 in HUVECs through bioinformatics prediction and experimental analysis. Based on previous research, miR-942-5p was selected as a potential target for circ_0003204. Several studies have corroborated that miR-942-5p plays a pro-oncogenic role in different cancers, including gastric carcinoma (32), cervical cancer (33), and lung cancer (34). A recent research demonstrated that ox-LDL treatment strikingly down-regulated miR-942 in endothelial cells and macrophages (18). Herein, we disclosed that miR-942-5p down-regulation abrogated the impact of circ_0003204 depletion on ox-LDLtriggered HUVECs damage.
Moreover, accumulating investigations have elaborated that circRNAs participate in many biological processes by indirectly regulating gene expression via competing with mRNA 3 ′ UTR for miRNA binding sites (35). Our report indicated that miR-942-5p could target HDAC9 by combining with its 3 ′ UTR. HDAC9 belongs to the class IIa histone deacetylases, which has a regulatory effect on the cardiovascular, musculoskeletal, nervous, and immune systems (36,37). The absence of HDAC9 hinders AS progression by reducing inflammation and reversing cholesterol transport (38). Malhotra et al. (39) reported that HDAC9 depletion blocked aortic calcification and increased VSMCs contractility. Han et al. (40) indicated that HDAC9 was overtly increased in ox-LDL-disposed endothelial cells, and its silencing hindered ox-LDL-triggered cell apoptosis and inflammation in endothelial cells. Consistently, we revealed that HDAC9 was prominently up-regulated in ox-LDL-disposed HUVECs. Moreover, miR-942-5p targeted HDAC9 to reduce ox-LDL-resulted HUVECs injury. Furthermore, we disclosed that circ_0003204 could modulate HDAC9 expression through decoying miR-942-5p.
In conclusion, circ_0003204 increased the expression of HDAC9 via sponging miR-942-5p, thereby preventing cell growth and promoting oxidative stress and inflammation in ox-LDL-mediated HUVECs. These findings demonstrated that circ_0003204 might be a promising therapeutic target for AS. The limitation of this work is the lack of in vivo experiments to verify the results of this research. In addition, more in-depth molecular mechanisms and their crosstalk with other studies need to be explored in future studies.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The First Affiliated Hospital of University of South China. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
HW: had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. HW and TY: study concept and design. TY and WL: acquisition of data. HW: critical revision of the manuscript for important intellectual content and study supervision. HW, TY, and WL: administrative, technical or material support. All authors: contributed to the article and approved the submitted version.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2021.646832/full#supplementary-material Supplementary Figure 1 | The expression levels of 6 candidate genes that might bind to miR-942-5p in HUVECs transfected with inhibitor NC or miR-942-5p inhibitor were detected by qPCR. Data were presented as mean ± SD, n = 3. * P < 0.05, and * * P < 0.01.