Hsa_circ_0003204 Knockdown Weakens Ox-LDL-Induced Cell Injury by Regulating miR-188-3p/TRPC6 Axis in Human Carotid Artery Endothelial Cells and THP-1 Cells

Background: Circular RNAs (circRNAs) are involved in atherosclerosis (AS) development. However, the function and mechanism of circRNA hsa_circ_0003204 (circ_0003204) in carotid artery AS remain unclear. Methods: Oxidized low-density lipoprotein (ox-LDL)-treated human carotid artery endothelial cells (HCtAECs) and THP-1 cells were used as cell models of carotid artery AS. Relative levels of circ_0003204, microRNA-188-3p (miR-188-3p), and transient receptor potential canonical channel 6 (TRPC6) were detected by quantitative reverse transcription–polymerase chain reaction or Western blotting. The targeting relationship between circ_0003204 or TRPC6 and miR-188-3p was assessed via dual-luciferase reporter analysis and RNA immunoprecipitation. Cell proliferation was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was analyzed via assessing cell caspase-3 activity, apoptosis, and apoptosis-related protein. Inflammatory response was analyzed via analysis of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Oxidative stress was assessed via determination of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Results: Circ_0003204 and TRPC6 levels were elevated, and miR-188-3p expression declined in ox-LDL-treated HCtAECs and THP-1 cells. Circ_0003204 could regulate TRPC6 expression via mediating miR-188-3p. Circ_0003204 silencing weakened ox-LDL-induced viability inhibition and apoptosis in HCtAECs, and inflammatory response and oxidative stress in THP-1 cells via regulating miR-188-3p. MiR-188-3p overexpression attenuated ox-LDL-induced injury in HCtAECs and THP-1 cells by targeting TRPC6. Conclusion: Circ_0003204 knockdown mitigated ox-LDL-induced injury in HCtAECs and THP-1 cells via regulating the miR-188-3p/TRPC6 axis, indicating that circ_0003204 might play an important role in carotid artery AS.


INTRODUCTION
Atherosclerosis (AS) is an inflammatory-related cardiovascular disease (1). Carotid artery AS is a group of AS and associated with the increased risk of cardiovascular disorders (2). Oxidized low-density lipoprotein (ox-LDL) has an essential role in AS development via regulating the function of multiple cell lines, like endothelial cells and macrophages (3). The malfunction of endothelial and THP-1 cells is implicated in the pathobiology of AS (4,5). Therefore, analyzing the pathogenesis of ox-LDLtriggered dysfunction of carotid artery endothelial cells and THP-1 cells may help to explore new strategies for carotid artery AS treatment.
Circular RNAs (circRNAs) and microRNAs (miRNAs) are correlated with the regulation of cardiovascular cell biology in AS (6). CircRNAs are stable ncRNAs formed via back-splicing events, which act as vital biomarkers for cardiovascular diseases, including AS (7). CircRNA hsa_circ_0003204 (circ_0003204), derived from ubiquitin-specific peptidase 36 (USP36), has been reported to be upregulated in ox-LDL-challenged human aortic endothelial cells (HAECs) and plays a vital role in cerebrovascular atherogenesis progression (8). Moreover, circ_0003204 is reported to be dysregulated in ox-LDL-irritated human umbilical vein endothelial cells (HUVECs) (9). Hence, we assumed that circ_0003204 might play a vital role in AS progression. However, how and whether circ_0003204 takes part in the development of carotid artery AS remain unknown.
The transient receptor potential canonical channels (TRPCs) play an important role in cardiovascular diseases (13). Among all miR-188-3p targets predicted by the DIANA tool, five mRNAs [KLF6, IGF2, AKT3, TRIM14, and TRPC 6 (TRPC6)] have been reported to have the opposite function of miR-188-3p. And preliminary experiments showed that TRPC6 was pulled down the most by the miR-188-3p probe. TRPC6, a key member of TRPCs, contributes to ox-LDL-induced HAEC apoptosis (14). At present, the involvement of circ_0003204, miR-188-3p, and TRPC6 in carotid artery AS is unclear. Hence, we hypothesized that circ_0003204 might regulate carotid artery AS development via mediating miR-188-3p/TRPC6 axis.
In this research, we utilized ox-LDL-challenged HCtAECs and THP-1 cells to mimic carotid artery AS environment (15). Moreover, we detected the expression of circ_0003204, miR-188-3p, and TRPC6, and we explored the function of circ_0003204 in ox-LDL-triggered cell injury. Additionally, we explored the ceRNA network of circ_0003204/miR-188-3p/TRPC6 axis.
To mimic the carotid AS-like microenvironment, HCtAECs and THP-1 cells were challenged via different doses of ox-LDL (Solarbio, Beijing, China) for 24 h.

Real-Time Quantitative PCR
The RNA was extracted by TRIzol (Thermo Fisher) (16). The RNA was reversely transcribed using a specific reverse transcription kit (Thermo Fisher). The cDNA was mixed with SYBR (Vazyme, Nanjing, China) and specific primers (Genscript, Nanjing, China) and used for qRT-PCR. The primers were as follows: circ_0003204 (F, . U6 or GAPDH served as a reference control. RNA level was computed using the 2 − Ct method (17).
The Magna RIP Kit (Sigma, St. Louis, MO, USA) was exploited for RIP analysis; 1 × 10 7 HCtAECs and THP-1 cells were lysed and interacted with anti-Ago2 or anti-IgG-conjugated magnetic beads for 6 h. The enrichment of circ_0003204, miR-188-3p, and TRPC6 in the complex was detected by qRT-PCR.
(Beyotime, Shanghai, China). The absorbance was examined at 570 nm using a microplate reader.

Caspase-3 Activity and Flow Cytometry
For detection of caspase-3 activity, 4 × 10 5 HCtAECs were added into 6-well plates and exposed to 100 µg/ml of ox-LDL. Next, cells were lysed for caspase-3 activity analysis using a caspase-3 assay kit (Abcam) according to the instruction of the manufacturer. For analysis of cell apoptotic rate, 2 × 10 5 HCtAECs were placed into 6-well plates overnight and then stimulated via 100 µg/ml of ox-LDL for 24 h. Next, cells were detected using Annexin V-FITC apoptosis detection kit (Sigma). The apoptotic cells were examined using a flow cytometer (Agilent, Hangzhou, China).

Enzyme-Linked Immunosorbent Assay
The inflammatory response was assessed via analysis of the levels of IL-1β, IL-6, and TNF-α; 1 × 10 5 THP-1 cells were placed into 12-well plates overnight and then treated via 100 µg/ml of ox-LDL for 24 h. Next, the medium was collected and used for analysis of IL-1β, IL-6, and TNF-α levels using specific ELISA kits (Thermo Fisher) following the instructions of the manufacturer.

Detection of Malondialdehyde, Superoxide Dismutase, and Reactive Oxygen Species Levels
The oxidative stress was analyzed by evaluation of malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) levels; 4 × 10 4 THP-1 cells were placed into 24-well plates overnight and then challenged via 100 µg/ml of ox-LDL for 24 h. Next, cells were lysed; and the lysis solution was used for analysis of MDA and SOD levels using MDA or SOD Assay kit (Sigma) following the instructions of the manufacturer. The levels of MDA and SOD were normalized to total protein in cells. In addition, 2 × 10 4 THP-1 cells were added into 96-well plates overnight and then treated via 100 µg/ml of ox-LDL for 24 h. Next, cells were analyzed using a cellular ROS assay kit (Abcam). The ROS level was normalized to the control group (not treated with ox-LDL).

Circ_0003204 and Transient Receptor
Furthermore, miR-188-3p level was progressively reduced as the elevation of ox-LDL (Figures 1C,D). Additionally, TRPC6 protein expression was markedly elevated in the two cell lines after exposure to different doses of ox-LDL (Figures 1E,F). These data indicated that circ_0003204, miR-188-3p, and TRPC might be associated with ox-LDLinduced injury.

Circ_0003204 Regulates Transient Receptor Potential Canonical Channel Expression via Mediating microRNA-188-3p
We further analyzed whether circ_0003204 could act as a ceRNA to regulate the miR-188-3p/TRPC axis. The predicted  binding sequences between miR-188-3p and circ_0003204 or TRPC are displayed in Figures 2A,B. To identify their interactions, the dual-luciferase reporter analysis was performed in HCtAECs and THP-1 cells transfected with circ_0003204-WT, circ_0003204-WT+miR-con, circ_0003204-WT+miR-188-3p mimic, circ_0003204-WT+miR-188-3p mimic+pcDNA, or TRPC overexpression vector. The luciferase activity of circ_0003204-WT evidently declined via miR-188-3p overexpression, which was impaired via introduction of TRPC6 (Figures 2C,D). Furthermore, the RIP analysis using anti-Ago2 revealed that circ_0003204, miR-188-3p, and TRPC were enriched in the same complex (Figures 2E,F). As exhibited in Figures 3A,B, transfection of the circ_0003204 overexpression vector led to an elevation in circ_0003204 level, whereas transfection of si-circ_0003204 led to a reduction in circ_0003204 level in both HCtAECs and THP-1 cells.

Transient Receptor Potential Canonical Channel 6 Knockdown Weakens Oxidized Low-Density Lipoprotein-Induced Injury in Human Carotid Artery Endothelial Cells and THP-1 Cells
To analyze the effects of TRPC6 knockdown on ox-LDLinduced injury in HCtAECs and THP-1 cells, we knocked out TRPC6 by transfection with si-TRPC6. The transfection efficiency is exhibited in Supplementary Figure 1A. Moreover, introduction of si-TRPC6 weakened the elevation of TRPC6 mediated by ox-LDL stimulation (Supplementary Figure 1B).
As expected, TRPC6 downregulation weakened ox-LDL-induced viability and proliferation inhibition (Supplementary Figures 1C-E). Moreover, the elevated caspase-3 activity, apoptotic rate, and BAX protein levels and the decreased BCL-2 protein levels in HCtAECs and THP-1 cells caused by ox-LDL were mitigated by TRPC6 knockdown (Supplementary Figures 1F-I). In addition, ox-LDL-induced cell inflammation (Supplementary Figures 1K-M) and oxidative stress (Supplementary Figures 1N-P) were impaired after TRPC6 silencing. These results suggested that TRPC6 silencing weakens ox-LDL-induced injury in HCtAECs and THP-1 cells.

DISCUSSION
AS causes high morbidity and mortality all around the world (18). Carotid artery AS is a common form of AS (19). The inflammatory response, oxidative stress, and endothelial cell apoptosis are implicated in AS progression (20)(21)(22). CircRNAs play a key role in the development of AS (23). This research focused on the function and mechanism of circ_0003204 in the regulation of ox-LDL-induced inflammatory response, oxidative stress, and endothelial cell apoptosis. Here, we first found that circ_0003204 knockdown could mitigate ox-LDL-induced injury in HCtAECs and THP-1 cells.
We established ox-LDL-stimulated HCtAECs and THP-1 cells, and we found that circ_0003204 expression was elevated in the two cell lines, which was consistent with that in HAECs or HUVECs (8,9). Hence, we assumed the increase in circ_0003204 induced by abnormal ox-LDL might be correlated with carotid artery AS development. Previous studies suggested that ox-LDL could induce endothelial cell injury and that ox-LDLstimulated HCtAECs could be used to assess the pathogenesis of carotid artery AS in vitro (15,24,25). Similarly, our study also found that ox-LDL caused HCtAEC viability inhibition and apoptosis promotion and that circ_0003204 knockdown weakened ox-LDL-exposed HCtAEC injury. In addition, ox-LDL-mediated THP-1 cell damage is also involved in the development of carotid artery AS (15,26,27). By detecting the pro-inflammatory cytokine levels and oxidative stress-related markers, we confirmed that circ_0003204 silencing attenuated ox-LDL-induced inflammatory response and oxidative stress in THP-1 cells. Collectively, inhibition of circ_0003204 played an inhibiting effect in ox-LDL-induced injury in AS.
Previous studies suggested that circRNA-mediated ceRNA network is the important mechanism in cardiovascular diseases and ox-LDL-induced injury (28,29). The former work has confirmed that circ_0003204 could act as a ceRNA to regulate the miR-370-3p/TGFβR2 axis (8). Here, we first explored the potential association among circ_0003204, miR-188-3p, and TRPC6. In addition, we found that circ_0003204 could positively regulate TRPC6 expression by binding to miR-188-3p, indicating that circ_0003204 might function as a ceRNA for miR-188-3p to mediate TRPC6. Our study found that miR-188-3p overexpression weakened ox-LDL-induced viability inhibition and apoptosis promotion in HCtAECs. Furthermore, we confirmed miR-188-3p weakened ox-LDL-induced inflammatory response in THP-1 cells, which was similar to that in a previous study (12). Besides, our results also displayed that miR-188-3p could mitigate ox-LDL-induced oxidative stress in THP-1 cells. These data indicated the protective function of miR-188-3p in ox-LDL-induced damage. Additionally, miR-188-3p knockdown reversed the effect of circ_0003204 silencing on ox-LDL-induced damage in the two cell lines, implying that circ_0003204 could regulate ox-LDL-induced cell injury by mediating miR-188-3p in HCtAECs and THP-1 cells.
Next, we validated the targeting interaction between miR-188-3p and TRPC6. Negri et al. and Thilo et al. reported that TRPC6 was abnormally expressed in vascular endothelial cells, and its elevation was associated with cardiovascular disease development (30,31). Moreover, Zhang et al. suggested that TRPC6 could contribute to AS development by promoting endothelial cell apoptosis (14). Similarly, our study also confirmed that the inhibitive role of miR-188-3p on ox-LDL-mediated cell injury in HCtAECs and THP-1 cells was partly reversed by TRPC6 overexpression. Collectively, we concluded that circ_0003204 could regulate ox-LDL-induced cell injury by mediating miR-188-3p and TRPC6. However, it did not indicate the biological role of circ_0003204 in carotid artery AS in vivo because of the alteration of microenvironment. The animal models have been widely used to assess the pathogenesis of AS in vivo (32). Therefore, the preclinical experiments using animals would be performed to test the function and mechanism of circ_0003204 in carotid artery AS in future.
In conclusion, circ_0003204 knockdown could attenuate ox-LDL-induced HCtAEC apoptosis and THP-1 cell inflammatory response and oxidative stress via modulating miR-188-3p/TRPC6 axis in a ceRNA network (Figure 8). This study indicated that this ceRNA crosstalk might be associated with ox-LDL-induced injury in carotid artery AS and that circ_0003204 might act as a target for carotid artery AS therapy.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.