Hsa_circ_0044097 Serves as a Promising Biomarker of Atherosclerosis and Its Effects on Vascular Smooth Cell Proliferation and Migration

ABSTRACT

Atherosclerosis (AS) is closely related to the pathogenesis and abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). To explore the value of hsa_circ_0044097 in the clinical management of AS and to elucidate its role and mechanism in the injury of VSMCs induced by in vitro oxidized low‐density lipoprotein (ox‐LDL). The mRNA expressions of hsa_circ_0044097 and miR‐3918 were determined using RT‐qPCR, and transfection verification was also conducted. The content of IL‐6 and TNF‐α was determined using an ELISA kit. CCK‐8 and transwell assays were performed to determine the proliferation and migration of HASMC cells. The dual luciferase reporter assay and Spearman correlation analysis were performed to verify the relationship between hsa_circ_0044097 and miR‐3918. The clinical value of hsa_circ_0044097 was evaluated using the ROC curve, K‐M survival analysis and Cox analysis. Hsa_circ_0044097 level was decreased in AS patients' serum and HASMCs stimulated by ox‐LDL. Hsa_circ_0044097 could serve as an indicator for the diagnosis of AS and for predicting the occurrence of MACE in AS patients. Overexpression of hsa_circ_0044097 inhibited the levels of inflammatory factors, cell proliferation and migration. MiR‐3918 was targeted by hsa_circ_0044097, and overexpression of miR‐3918 reversed the inhibitory effect of the upregulation of hsa_circ_0044097 on the proliferation and migration of VSMCs. Hsa_circ_0044097 may be a biomarker for diagnosing AS and predicting the occurrence of MACE. Hsa_circ_0044097 influences the progression of AS.

Hsa_circ_0044097 expression level was decreased AS patients and in ox‐LDL‐HASMC. The expression level of hsa_circ_0044097 is negatively correlated with the levels of hs‐CRP and LDL‐C. Hsa_circ_0044097 can be a biomarker for the diagnosis and prediction of MACE. Hsa_circ_0044097 sponging miR‐3918, regulating the proliferation and migration of ox‐LDL‐HASMCs and thereby influencing AS.

1. Introduction

The biggest killer on the planet is cardiovascular disease, which puts people's lives in danger [1]. Atherosclerosis (AS) is the key pathological basis of cardiovascular diseases. The occurrence of this disease is a complex pathological and physiological process, involving multiple processes such as endothelial damage, lipid infiltration, oxidative stress, inflammatory response, cell proliferation and migration [2]. Epidemiological data show that the annual mortality rate of AS‐related diseases in China is 230 per 100,000, and the incidence is showing a trend of becoming younger. As atherosclerosis progresses, the vascular lumen of patients will gradually become narrowed, leading to local ischemia [3]. Although surgical interventional treatments such as percutaneous transluminal angioplasty (PTA) can restore a certain blood flow to tissues and organs, over time, some of the opened blood vessels will again become narrowed [4]. Therefore, the management and treatment of AS has always been a highly challenging task. At present, the diagnosis of clinical AS mainly relies on ultrasound to detect plaque burden and serum LDL‐C levels. However, this method has the drawbacks of insufficient sensitivity and inability to assess plaque activity. Therefore, there is an urgent need in clinical practice to discover new molecular markers for the diagnosis of AS.

Recent findings have revealed that circular RNAs (circRNAs) are involved in the AS process, regulating the phenotypic transformation of VSMCs [5]. CircRNAs exhibit tissue‐specific expression patterns, and their stability and sponge adsorption properties make them an ideal candidate for disease biomarkers [6]. For instance, increasing the expression of circRNA‐LONP2 can promote endothelial inflammation and atherosclerosis [7]. CircARCN1 is mainly expressed in monocytes and macrophages. Upregulation of circARCN1 expression promotes the progression of atherosclerosis [8]. CircRNA circEsyt2 was upregulated during vascular remodeling, and it enhanced proliferation and migration and inhibited apoptosis and differentiation in VSMCs [9]. However, the relationship and mechanism of action between hsa_circ_0044097 and AS have not yet been reported.

This study aims to explore the value of hsa_circ_0044097 in the diagnosis and clinical management of AS, as well as its potential mechanism of action in vascular smooth muscle cells (HASMC). RT‐qPCR revealed that the serum of AS patients and ox‐LDL‐HASMC contained elevated levels of hsa_circ_0044097. The cell function experiments confirmed that hsa_circ_0044097 regulates the development of AS by influencing the cell proliferation and migration of HASMC.

2. Materials and Methods

2.1. Clinical Patients and Samples

A total of 113 patients diagnosed with asymptomatic AS is in Donghai County People's Hospital of Lianyungang from February 2024 to October 2024 were recruited. In addition, a control group of 85 healthy volunteers who underwent physical examinations at our hospital was randomly selected. The study was reviewed and approved by the Ethics Committee of Donghai County People's Hospital of Lianyungang, and informed consent was obtained from all patients. All participants underwent the relevant physical examinations. We collected their age, gender, Body Mass Index (BMI), Fasting Blood Glucose‌ (FBG), High‐Density Lipoprotein (HDL), Low‐Density Lipoprotein Cholesterol (LDL‐C), High‐sensitivity C‐reactive Protein (hs‐CRP), history of hypertension and diabetes, etc., as clinical data in Table 1.

Table 1.

The relationship between clinical data and the expression level of hsa_circ_0044097.

Paraments	total	hsa_circ_0044097 expression		p
		High (n = 56)	Low (n = 57)
Age				0.636
≥ 50	55	26	29
< 50	58	30	28
Sex				0.074
Male	58	24	34
Female	55	32	23
BMI (kg/m2)				0.396
≥ 23.9	55	25	30
< 23.9	58	31	27
FBG (mmol/L)				0.157
≥ 6.1	54	23	31
< 6.1	59	33	26
HDL (mmol/L)				0.507
< 1.3	54	25	29
≥ 1.3	59	31	28
LDL‐C (mmol/L)				0.048
≥ 3.4	55	22	33
<3.4	58	34	24
hs‐CRP (mg/L)				0.019
≥ 1.0	58	35	23
< 1.0	55	21	34
Hypertension				0.157
Yes	54	23	31
No	59	33	26
Diabetes				0.486
Yes	44	20	24
No	69	36	33

Abbreviations: BMI, Body Mass Index; FBG, Fasting Blood Glucose; HDL, High Density Lipoprotein; LDL‐C, Low‐Density Lipoprotein Cholesterol; hs‐CRP, High‐sensitivity C‐reactive Protein.

Inclusion criteria: (1) Patients were first diagnosed with AS. (2) Patients had complete clinical data. (3) Patients and their family members were informed of the purpose of the trial and possible medical risks, and their consent was obtained. Exclusion Criteria: (1) Patients with important organ dysfunction. (2) Patients with intracranial hemorrhage or bleeding tendency. (3) Patients had a malignant tumor and severe hepatic and renal insufficiency. (4) Patients had mental or speech disorders.

2.2. Follow‐Up

A 60‐month follow‐up record was conducted for patients with AS. Follow‐up records were kept via outpatient review appointments, telephone calls and video calls. The primary endpoint was Major Adverse Cardiovascular Events (MACE), including cardiovascular death, non‐fatal myocardial infarction, non‐fatal stroke, unstable angina, coronary syndrome requiring urgent revascularisation, and hospitalisation for heart failure.

2.3. Bioinformatics Process

The GSE152280 dataset is from the online database GEO (https://www.ncbi.nlm.nih.gov/geo/). This dataset analyzed the differential expression of circRNAs in 3 normal arteries and 3 atherosclerotic arteries. The target genes of miR‐3918 were predicted using the miRDB (https://mirdb.org/) and starBase (https://rnasysu.com/encori/) databases, and the targets related to AS diseases were retrieved from the GeneCards database simultaneously. Finally, the Venny 2.1.0 online data platform (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to draw the Venn diagram of the three.

2.4. RNA Stability

Regarding the RNase R digestion test, after extracting an equal amount of total RNA, the samples were treated under conditions with or without 3 U/μl RNase R (10109134001, MeRCK, Darmstadt, Germany). Then, they were incubated for 30 min. For the actinomycin D test, the cells were treated with 2 mg/mL actinomycin D (A9415‐25MG, MeRCK, Darmstadt, Germany) for 0 h, 4 h, 8 h, and 12 h. Finally, qRT‐PCR was used to evaluate the expression levels of hsa_circ_0044097 and EFTUD2.

2.5. Cell Culture

HASMCs was purchased from ChemicalBook. The cells were cultivated in a DEME (high glucose) medium with 10% FBS, 1% penicillin/streptomycin, and 1% vascular smooth muscle cell growth factor. The cultivation conditions are 37°C and 5% CO₂. When the cell density reaches 80%–90%, perform digestion and passage with trypsin. Use the cells that have been passaged for 4–8 generations for the subsequent experiments.

2.6. Constructing an AS Model by ox‐LDL‐Induced HASMC

The ox‐LDL was diluted to a final concentration of 50 μg/mL using the culture medium. Ox‐LDL was added to the cells when the cell density reaches 80%. And the culture is continued for 48 h. Finally, under the inverted microscope, it was observed that the HASMC changed from a long, spindle‐shaped contracted state to a round or irregular expanded state.

2.7. Cell Transfection

Transfect ox‐LDL‐induced HASMC cells with 1 μg/μL pcDNA3.1‐hsa_circ_0044097 and the pcDNA3.1 vector (ThermoFisher, USA). During transfection, follow the manufacturer's instructions to use Lipofectamine 3000 (ThermoFisher, USA) for the operation. In addition, miR‐3918 mimics and inhibitors (ThermoFisher, USA) were transfected into HASMCs induced by ox‐LDL.

2.8. RT‐qPCR

We extracted total RNA from the nuclear cells in the serum using the TRIzol reagent (ThermoFisher, USA). Using the mRNA reverse transcription kit (Qiagen), total RNA was reverse‐transcribed into cDNA. GAPDH and U6 were used as the reference gene for hsa_circ_0044097 and miR‐3918. The qRT‐PCR was performed on the Q2000B fluorescence quantitative PCR instrument. The relative expression was calculated using the 2^{− △△Ct} algorithm.

2.9. Western Blot

SDS‐PAGE was used to separate an equal amount of proteins. Then the separated proteins were transferred onto a PVDF membrane (3010040001, MeRCK, Darmstadt, Ger.) After blocking with non‐fat milk, the membranes were incubated overnight at 4°C with primary antibodies against PCNA (1:1000, HY‐P80268, MCE, Shanghai, China), Cyclin D1 (1:500, HY‐P80633, MCE, Shanghai, China), MMP9 (1:500, HY‐P80756, MCE, Shanghai, China), osteopontin (OPN) (1:500, HY‐P86670, MCE, Shanghai, China) and GAPDH (1:10000, HY‐P80137, MCE, Shanghai, China). After washing with TBST, the membranes were incubated with the secondary antibody at 37°C for 2 h. Finally, digital images were analyzed using ImageJ Software.

2.10. ELISA

We used an ELISA kit (MSK, Wuhan, China) to measure the levels of IL‐6 and TNF‐α. On the enzyme‐labeled plate pre‐coated with antibodies, set the standard sample wells, sample wells and blank wells. Add 100 μL of the standard or the test sample to each well, cover with a sealing film, and incubate at 37°C for 1.5–2 h. A total of 100 μL of biotin‐labeled detection antibody working solution was added. After incubation at 37°C for 1 h, 100 μL of TMB substrate solution was added to each well. After incubation at 37°C in the dark for 30 min, 50 μL of the termination solution was added to each well. Finally, the OD value was measured at 450 nm.

2.11. Cell Proliferation

The next step was to seed the transfected cells in 96‐well plates at a density of 1.5 × 10³ cells per well. Then, the CCK‐8 kit (ThermoFisher, USA) was used to detect the cell proliferation at 0, 24, 48, and 72 h. During the assay, remove the relevant 96‐well plate. Add 10 μL of CCK‐8 working solution to each well in the dark, shaking the plate to mix. Avoid air bubbles, which may affect the results. Return the plate to the incubator and incubate in the dark for the appropriate duration. Use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

2.12. Cell Migration Assay

In the logarithmic growth phase, the HASMC cells induced by ox‐LDL were cultured and then digested with trypsin. Resuspend the cells in fresh serum‐free DEME medium and adjust the cell concentration to 1 × 10⁶/mL. The cell suspension should be added to the upper chamber (100 μL), and 500 μL of DMEM medium containing 10% serum should be added to the lower chamber. Finally, the cells were fixed, stained and counted.

2.13. Luciferase Activity

The binding sites of hsa_circ_0044097 and miR‐3918 were predicted using the starBase database. Expand hsa_circ_0044097 and the CBS wild‐type sequence that contains the complete binding site of miR‐3918. They are inserted into the fluorescent reporter vector to construct the wild‐type plasmid of hsa_circ_0044097 (circ‐WT and CBS‐WT); at the same time, the core base of the binding site is subjected to site‐directed mutation to disrupt the complementary binding structure without changing the sequences of the remaining bases. The mutant plasmids of hsa_circ_0044097 and CBS (circ‐MUT and CBS‐MUT) are constructed.

2.14. Statistical Analysis

The data were processed and analyzed using SPSS 25.0 and Graphpad Prism 10.3.1. Quantitative data that followed a normal distribution were expressed as x ± s, and inter‐group comparisons were conducted using the t‐test. Multivariate Cox regression analysis was employed to investigate the influencing factors of the prognosis of AS patients. ROC curves were performed to analyze the value of the diagnosis of hsa_circ_0044097 in AS patients. A difference was considered statistically significant when p < 0.05.

3. Results

3.1. Hsa_circ_0044097 Expression Is Downregulated in AS and ox‐LDL‐Induced HASMCs

In order to study the impact of circRAN on the AS process, we mined the GSE152280 dataset from the GEO database. We used a p‐value < 0.05 as the cutoff threshold to screen for differentially expressed circRNAs in the three normal arteries and the three atherosclerotic arteries. Then, we used the WeiShengXin online platform (https://www.bioinformatics.com.cn/) to create a volcano plot for the differentially expressed genes in the GSE152280 dataset (Figure 1A). It was found that there were 948 upregulated circRNAs in the AS group, and 284 downregulated ones (Figure 1A). Among all the down‐regulated circRNAs, hsa_circ_0044097 has the largest log2(Fold Change) (Figure 1A). We speculate that this gene may have a significant impact on the AS. Therefore, hsa_circ_0044097 was chosen as the gene to study. Treatment with RNase‐R confirmed the strong resistance of hsa_circ_0044097 to degradation, whereas linear EFTUD2 mRNA was almost completely degraded (Figure 1B), confirming the circular nature of hsa_circ_0044097. Treatment with actinomycin D demonstrated that hsa_circ_0044097 exhibits greater stability and a longer half‐life in comparison to linear EFTUD2 mRNA (Figure 1C). RT‐qPCR was performed to measure hsa_circ_0044097 levels in the two groups of participants' serum. This gene was downregulated in AS patients (Figure 1D). The way in which hsa_circ_0044097 affects AS is explained further here. An AS cell model was established by stimulating HASMC cells with an ox‐LDL concentration of 50 μg/mL for 48 h. The ELISA results indicated that inflammatory factors (IL‐6 and TNF‐α) levels in the ox‐LDL‐HASMC group were abnormally elevated (Figure 1E). This indicates that we have successfully established an AS cell model induced by ox‐LDL. Furthermore, hsa_circ_0044097 expression was downregulated in the ox‐LDL‐HASMC group (Figure 1F). These results indicate that hsa_circ_0044097 plays a crucial role in AS.

Figure 1.

Hsa_circ_0044097 expression is downregulated in AS and ox‐LDL‐induced HASMCs. (A) Volcano plot showed the differentially expressed circRNAs between 3 normal arteries and 3 atherosclerotic arteries. (B) RT‐qPCR detected hsa_circ_0044097 and EFTUD2 mRNA level with or without RNase R treatment. (C) RT‐qPCR detected hsa_circ_0044097 and EFTUD2 mRNA level treated with actinomycin D. (D) Hsa_circ_0044097 was downregulated in AS patients. (E) The ELISA method was used to detect the levels of IL‐6 and TNF‐α in HASMCs induced by ox‐LDL. (F) Hsa_circ_0044097 was downregulated in HASMCs induced by ox‐LDL. ***p < 0.001.

3.2. The Relationship Between hsa_circ_0044097 and hs‐CRP and LDL‐C

As is well known, hs‐CRP is the core indicator of vascular inflammation, and LDL‐C is a specific marker for myocardial injury. Therefore, we conducted a statistical analysis of hs‐CRP and LDL‐C for the two groups of participants. The results indicated that the hs‐CRP level in AS patients was elevated, and it was significantly negatively correlated with hsa_circ_0044097 (r = −0.6249, p < 0.001) (Figure 2A, B). Similarly, LDL‐C expression level also showed the same trend. The level of LDL‐C in patients with AS was also significantly increased, and it was also significantly negatively correlated with hsa_circ_0044097 (r = −0.5550, p < 0.001) (Figure 2C, D). These results suggest that hsa_circ_0044097 could be a biomarker to help diagnose and manage AS. However, LDL‐C (p = 0.048) and hs‐CRP (p = 0.019) were correlated with hsa_circ_0044097 expression level.

Figure 2.

The relationship between hsa_circ_0044097 and hs‐CRP and LDL‐C. (A) The level of hs‐CRP was elevated in AS patients. (B) Hsa_circ_0044097 was negatively correlated with the content of hs‐CRP. (C)The level of LDL‐C was elevated in the AS patients. (D) Hsa_circ_0044097 was negatively correlated with the content of LDL‐C. ***p < 0.001.

3.3. The Clinical Value of hsa_circ_0044097

We used the average of hsa_circ_0044097 mRNA level as the cutoff threshold, and divided the AS patients into the hsa_circ_0044097 high‐expression group (n = 56) and the hsa_circ_0044097 low‐expression group (n = 57). The relationship between the clinical data of AS patients and hsa_circ_0044097 expression level was analyzed using the chi‐square test. As shown in Table 1, the Age (p = 0.636), Sex (p = 0.074), BMI (p = 0.396), FBG (p = 0.157), HDL (p = 0.507), Hypertension (p = 0.157) and Diabetes (p = 0.486) of the patients were not significantly associated with hsa_circ_0044097. In addition, we conducted an ROC analysis on the hsa_circ_0044097 expression levels in healthy controls and AS patients. The results indicated that the AUC of this ROC curve was 0.9083, with a 95% CI of 0.8706–0.9460, and the sensitivity of this curve was 85.9% and the specificity was 75.20% (Figure 3A). This reveals that hsa_circ_0044097 expression level can effectively distinguish healthy participants from AS patients. Furthermore, in the study, we performed a 60‐month follow‐up report on all AS patients. The Major Adverse Cardiovascular Events‌ (MACE) that occurred during this period are defined as the endpoint events. The results of the K‐M analysis indicated that the low‐expression hsa_circ_0044097 patients had a higher likelihood of experiencing MACE (Figure 3B). The results of the multivariate Cox analysis also indicated that hsa_circ_0044097 could independently serve as an indicator for predicting the occurrence of major adverse cardiovascular events (MACE) in patients with AS Table 2.

Figure 3.

The clinical value of hsa_circ_0044097. (A) ROC curve of hsa_circ_0044097. (B) K‐M analysis predicts the incidence rate of MACE in AS patients. ***p < 0.001.

Table 2.

Multivariate Cox analysis to evaluate the predictive value of hsa_circ_0044097 for AS.

	HR	95% CI	P
hsa_circ_0044097	0.393	0.165–0.935	0.035
Age	1.222	0.546–2.737	0.625
Sex	1.128	0.502–2.534	0.770
BMI (kg/m2)	1.302	0.589–2.880	0.515
FBG (mmol/L)	1.597	0.720–3.543	0.249
HDL (mmol/L)	0.893	0.402–1.985	0.781
LDL (mmol/L)	1.131	0.498–2.571	0.769
hs‐CRP (mg/L)	1.348	0.571–3.184	0.496
Hypertension	1.227	0.537–2.804	0.627
Diabetes	1.284	0.560–2.947	0.555

Abbreviations: BMI, Body Mass Index; FBG, Fasting Blood Glucose; HDL, High Density Lipoprotein; LDL‐C, Low‐Density Lipoprotein Cholesterol; hs‐CRP, High‐sensitivity C‐reactive Protein.

3.4. Hsa_circ_0044097 Affects the Proliferation and Migration of HASMCs

The investigation will explore the way in which hsa_circ_0044097 impacts AS. hsa_circ_0044097 was overexpressed in ox‐LDL‐HASMC cells. The RT‐qPCR results showed that the hsa_circ_0044097 in the OE‐circRNA group level was higher than the vector group (Figure 4A). Overexpression of hsa_circ_0044097 reduced the levels of IL‐6 and TNF‐α in ox‐LDL‐HASMC cells (Figure 4B). Overexpression of hsa_circ_0044097 restrains the proliferation of ox‐LDL‐HASMC cells (Figure 4C). At the same time, it was observed that the overexpression of hsa_circ_0044097 inhibited the protein expression levels of PCNA and Cyclin D1 (Figure 4D). Furthermore, overexpression of hsa_circ_0044097 inhibited the migration of ox‐LDL‐HASMC cells (Figure 4E). Similarly, overexpression of hsa_circ_0044097 inhibited the protein levels of MMP‐9 and OPN (Figure 4F).

Figure 4.

Hsa_circ_0044097 affects the proliferation and migration of HASMCs. (A) RT‐qPCR was used to verify the transfection effect of pcDNA3.1‐hsa_circ_0044097. (B) Overexpression of hsa_circ_0044097 inhibits the levels of IL‐6 and TNF‐α in HASMCs induced by ox‐LDL. (C) Overexpression of hsa_circ_0044097 inhibited the cell proliferation of HASMCs induced by ox‐LDL. (D) Overexpression of hsa_circ_0044097 inhibited PCNA and Cyclin D1 expression in HASMCs cells induced by ox‐LDL. (E) Overexpression of hsa_circ_0044097 inhibited the cell migration of HASMCs induced by ox‐LDL. (F) Overexpression of hsa_circ_0044097 inhibited MMP‐9 and OPN expression in HASMCs cells induced by ox‐LDL. **p < 0.01, ***p < 0.001.

3.5. Hsa_circ_0044097 Affects the Proliferation and Migration of HASMCs by miR‐3918

To further clarify the specific mechanisms by which hsa_circ_0044097 affects the inflammatory response, proliferation, and migration of ox‐LDL‐HASMC cells, we used the starBase database to predict the downstream target miRNAs of hsa_circ_0044097. We found that miR‐3918 and hsa_circ_0044097 have a relatively high TDMDScore. Furthermore, there are reports indicating that miR‐3918 is upregulated in Primary Central Nervous System Lymphoma (PCNSL) [10]. Therefore, we chose miR‐3918 as our research subject. We found that miR‐3918 was upregulated in AS patients (Figure 5A). Furthermore, the luciferase activity of the circRNA‐WT group was inhibited by the miR‐3918 mimics, but there was no significant effect on the circRNA‐MUT group (Figure 5B). Interestingly, miR‐3918 inhibitors instead exhibited the opposite effect (Figure 5B). Additionally, the RIP experiment demonstrated that, compared with the Anti‐IgG group, both hsa_circ_0044097 and miR‐3918 genes were significantly enriched in the Anti‐Ago2 group (Figure 5C). Furthermore, the correlation analysis indicated that the expression level of miR‐3918 had a significant negative regulatory relationship with hsa_circ_0044097, with r = −0.5420 and p < 0.001 (Figure 5D). Next, pcDNA‐ hsa_circ_0044097 and miR‐3918 mimics were co‐transfected into ox‐LDL‐HASMC cells. It was found that the miR‐3918 mimic reversed the inhibitory effect of overexpressing hsa_circ_0044097 on miR‐3918 level (Figure 5E). The cell function experiments revealed that the miR‐3918 mimics restored the inhibitory effect of overexpressed hsa_circ_0044097 on IL‐6 and TNF‐α (Figure 5F). The inhibitory effect on the proliferation and migration of ox‐LDL‐HASMC cells of the overexpressed hsa_circ_0044097 was reversed by the miR‐3918 mimics, similarly (Figure 5G–J).

Figure 5.

Hsa_circ_0044097 affects the proliferation and migration of HASMCs by miR‐3918. (A) MiR‐3918 expression was upregulated in AS patients. (B) The effect of transfection with miR‐3918 mimics/inhibitors on the luciferase activity of circ‐WT and circ‐MUT. (C) The RIP experiment verified the interaction between hsa_circ_0044097 and miR‐3918 in cells. (D) The Spearman correlation analysis for hsa_circ_0044097 and miR‐3918. (E) The transfection effects of pcDNA3.1‐hsa_circ_0044097 and miR‐3918 mimics were verified by RT‐qPCR. (F) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the inflammatory factors (IL‐6 and TNF‐α) induced by ox‐LDL in HASMCs. (G) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the proliferation induced by ox‐LDL in HASMCs. (H) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the PCNA and Cyclin D1 expression induced by ox‐LDL in HASMCs. (I) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the migration induced by ox‐LDL in HASMCs. (J) The miR‐3918 mimic reversed the inhibitory effect of overexpressed hsa_circ_0044097 on the MMP‐9 and OPN expression induced by ox‐LDL in HASMCs. ***p < 0.001.

3.6. Hsa_circ_0044097 Affects the Proliferation and Migration of HASMCs Through the miR‐3918/CBS Axis

As shown in the Venn diagram, CBS is the only target gene of miR‐3918 predicted by the databases and associated with AS (Figure 6A). The RT‐qPCR results indicated that CBS was significantly downregulated in the serum of AS patients (Figure 6B). The dual‐luciferase assay demonstrated that there is an interaction between miR‐3918 and CBS (Figure 6C). The RIP experiment indicated that the Ago2 protein has a binding association with CBS and miR‐3918, suggesting that miR‐3918 may regulate the expression of CBS by binding to the Ago2 protein (Figure 6D). Furthermore, the correlation analysis showed that CBS was negatively correlated with miR‐3918, with r = −0.8055 and p < 0.001 (Figure 6E). Subsequently, pcDNA‐hsa_circ_0044097, miR‐3918 mimics and pcDNA‐CBS were successfully co‐transfected into ox‐LDL‐HASMC cells (Figure 6F). Upregulation of CBS reversed the promoting effect of co‐overexpression of hsa_circ_0044097 and miR‐3918 on the inflammatory response (Figure 6G). The CCK‐8 results showed that the upregulation of CBS reversed the promoting effect of co‐overexpression of hsa_circ_0044097 and miR‐3918 on cell proliferation (Figure 6H). The Western blot results for PCNA and Cyclin D1 further support the regulatory role of CBS in the proliferation of ox‐LDL‐HASMC cells (Figure 6I). The final transwell results indicated that the upregulation of CBS reversed the promoting effect of the co‐overexpression of hsa_circ_0044097 and miR‐3918 on cell migration (Figure 6J). Similarly, the Western blot results of MMP‐9 and OPN further confirmed the regulatory effect of CBS on the migration of ox‐LDL‐HASMC cells (Figure 6K).

Figure 6.

Hsa_circ_0044097 affects the proliferation and migration of HASMCs through the miR‐3918/CBS axis. (A) Venn diagram of the downstream target genes of miR‐3918. (B) CBS expression was downregulated in AS patients. (C) The effect of transfection with miR‐3918 mimics/inhibitors on the luciferase activity of CBS‐WT and CBS‐MUT. (D) The RIP experiment verified the interaction between CBS and miR‐3918 in cells. (E) The Spearman correlation analysis for CBS and miR‐3918. (F) The transfection effects of pcDNA3.1‐hsa_circ_0044097, miR‐3918 mimics, and pcDNA3.1‐CBS were verified by RT‐qPCR. (G) The ELISA kit measured the levels of inflammatory factors (IL‐6 and TNF‐α). (H) The CCK‐8 method evaluated the proliferation ability of cells. (I) Western blot was used to detect the protein expression levels of PCNA and Cyclin D1. (J) The Transwell method evaluated the migration ability of cells. (K) Western blot was used to detect the protein expression levels of MMP‐9 and OPN. ***p < 0.001.

4. Discussion

AS is the main pathological basis of cardiovascular diseases, characterized by lipid deposition, chronic inflammatory response, and fibrous plaque formation in the vascular wall [11, 12]. Recent studies have shown that epigenetic regulation, especially the abnormal expression of circRNAs, is crucial in the progression of AS [9, 13]. The key pathological processes of AS include endothelial cell dysfunction, imbalance in macrophage polarization, and phenotypic transformation of VSMCs [14]. Among them, the abnormal transformation of VSMCs from a contractile state to a synthetic state significantly promotes the instability of the plaque [15]. However, the specific mechanism by which hsa_circ_0044097 affects the progression of AS through influencing the proliferation and migration of VSMCs still needs to be further elucidated. In this study, we analyzed the GSE152280 dataset from the GEO database and observed that hsa_circ_0044097 was downregulated in this dataset. Meanwhile, through the execution of RT‐qPCR, we observed that hsa_circ_0044097 was downregulated in AS. We successfully established an atherosclerotic cell model (ox‐LDL‐HASMCs) by stimulating HASMC cells with 50 μg/mL of ox‐LDL. We observed that hsa_circ_0044097 expression level was also downregulated in ox‐LDL‐HASMCs.

The circular structure of circRNAs endows them with stability against RNA degradation, making them ideal biomarkers for liquid biopsy. It can regulate downstream target genes through the competitive endogenous RNA (ceRNA) mechanism. As we know, LDL‐C and hs‐CRP are crucial biomarkers for diagnosing AS in clinical practice [16, 17]. They respectively represent lipid metabolism disorders and inflammatory states, and jointly drive the progression of AS. LDL‐C is the main lipid risk factor for atherosclerosis [18]. High levels of LDL‐C can penetrate the vascular endothelium, undergo oxidative modification within the vessel wall, trigger the infiltration of monocytes to form foam cells, and simultaneously promote plaque formation and vascular narrowing [19]. And hs‐CRP is a sensitive indicator of inflammation [20]. It can promote the adhesion of monocytes to the vascular endothelium, enhance the transformation of macrophages into foam cells, stimulate pro‐inflammatory factors release, thereby accelerating the instability and rupture of plaques [21]. This study found that both LDL‐C and hs‐CRP were significantly elevated in AS patients, and hsa_circ_0044097 expression level was negatively correlated with both of them. This result suggests that hsa_circ_0044097 has the potential to serve as a biomarker for AS. Subsequently, we used the ROC curve, K‐M analysis and COX analysis to evaluate the clinical value of hsa_circ_0044097 in the treatment of AS. The results show that the hsa_circ_0044097 ROC curve can effectively distinguish healthy controls from AS patients. Furthermore, the K‐M analysis revealed that patients with low expression of hsa_circ_0044097 always had a higher incidence of MACE. Finally, the COX result indicated that hsa_circ_0044097 could serve as an independent predictor for whether patients with AS would develop MACE.

The abnormal proliferation and migration of VSMCs are the key factors leading to the thickening of the arterial intima [15]. Studies have shown that VSMCs that migrate to the intima will engulf ox‐LDL, then transform into myogenic foam cells, and together with macrophage‐derived foam cells, form a lipid core [22]. Meanwhile, the excessive proliferation of VSMCs will lead to an increase in plaque size and narrowing of the lumen [23]. Furthermore, in a chronic inflammatory environment, VSMCs will transform into osteoblast‐like cells, secreting calcium, which deposits in the plaque, thereby accelerating the hardening and brittleness of the plaque [24]. A growing body of research has shown that during the progression of AS, circRNAs play a significant role in the behavior of VSMCs. For instance, promoting the proliferation and angiogenesis ability of HUVECs can be achieved by inhibiting the expression of hsa_circ_0003575 [25]. Inhibiting the expression of hsa_circ_0030042 reduced the proliferation and migration of VSMCs induced by TNF‐α, while increasing cell apoptosis [26]. Our research has found that overexpression of hsa_circ_0044097 can reduce the levels of IL‐6 and TNF‐α in ox‐LDL‐HASMC. Our research has found that overexpression of hsa_circ_0044097 can reduce the levels of IL‐6 and TNF‐α in ox‐LDL‐HASMC. At the same time, it inhibits its proliferation and migration. The way in which hsa_circ_0044097 affects the proliferation and migration of VSMCs still needs to be clarified. We used the starBase database to predict its downstream target miR‐3918. At the same time, through the dual luciferase reporter assay and Spearman correlation analysis, we demonstrated the negative regulatory relationship between hsa_circ_0044097 and miR‐3918. It is reported that hsa_circ_0001402 targets miR‐183‐5p to increase the level of FKBPL, thereby suppressing ‌the proliferation and migration of VSMCs [27]. Another report indicated that CircUBR4B targets miR‐185‐5p and affects the proliferation and migration of VSMCs induced by ox‐LDL [28]. It is worth noting that our research results are similar to those reported above. Our experimental results indicate that the miR‐3918 mimics reversed the inhibitory effects of overexpressed hsa_circ_0044097 on IL‐6 and TNF‐α, cell proliferation, and migration of ox‐LDL‐HASMC.

5. Conclusion

Hsa_circ_0044097 expression level was decreased in AS patients and in ox‐LDL‐HASMC. The expression level of hsa_circ_0044097 is negatively correlated with the levels of hs‐CRP and LDL‐C. Hsa_circ_0044097 can be a biomarker for the diagnosis and prediction of MACE. Hsa_circ_0044097 sponging miR‐3918, regulating the proliferation and migration of ox‐LDL‐HASMCs and thereby influencing AS.

Author Contributions

Cencen Ren: conceptualization, data curation, formal analysis, software, writing – original draft. Yungen Jiao: conceptualization, methodology, supervision, validation, writing – review and editing.

Funding

The authors have nothing to report.

Ethics Statement

This study was performed in line with the principles of the Declaration of Helsinki. The study was reviewed and approved by the Ethics Committee of Donghai County People's Hospital of Lianyungang.

Consent

Informed consent was obtained from all patients.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.