Circ_CHMP5 aggravates oxidized low-density lipoprotein-induced damage to human umbilical vein endothelial cells through miR-516b-5p/TGFβR2 axis

Abstract

BACKGROUND:

Atherosclerosis (AS) was one of the main causes of death in the elderly, and lesions in human umbilical vein endothelial cells (HUVECs) could lead to AS. CircRNA-charged multivesicular body protein 5 (circ_CHMP5) was reported to participate in the progression of AS.

METHODS:

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the levels of circ_CHMP5, miR-516b-5p, and transforming growth factor beta receptor 2 (TGFβR2) in AS patients or ox-LDL-induced HUVECs. 5-Ethynyl-2’-deoxyuridine and cell counting kit-8 assays were performed to detect cell proliferation. Proteins expression was assessed by western blot assay. Cell apoptosis was examined by flow cytometry. Tube formation assay was utilized to measure the tube formation ability of HUVCEs. The targeting relationships between miR-516b-5p and circ_CHMP5 or TGFβR2 were confirmed by dual-luciferase reporter assay and RNA-pull down assay.

RESULTS:

Circ_CHMP5 was enhanced in the serum of AS patients and ox-LDL-exposure HUVECs. Ox-LDL blocked proliferation and tube formation of HUVECs and induced cell apoptosis, and circ_CHMP5 knockdown reversed these effects. In addition, circ_CHMP5 regulated the growth of ox-LDL-induced HUVECs through miR-516b-5p and TGFβR2. Moreover, the effects of circ_CHMP5 knockdown on ox-LDL-induced HUVECs were obviously recovered by downregulation of miR-516b-5p, and overexpression of TGFβR2 restored the effects of miR-516b-5p upregulation on ox-LDL-stimulated HUVECs.

CONCLUSION:

Silence of circ_CHMP5 overturned ox-LDL-treated inhibition of HUVECs proliferation and angiogenesis by miR-516b-5p and TGFβR2. These results provided new solutions for the treatment of AS.

Keywords

Circ_CHMP5 miR-516b-5p TGFβR2 HUVECs AS

1 Introduction

Atherosclerosis (AS) is a disease characterized by atherosclerosis that occurs in the arteries of the human body [1]. It occurs in the elderly and is related to hypertension, hyperlipidemia, and ox-LDL [2, 3], which can cause injury of arterial endothelial cells and further lead to AS [4]. AS is the most common cause of death all over the world [5, 6], so finding the genetic basis and molecular target of AS by molecular means is of great importance for the diagnosis and treatment of AS.

Previous studies had shown that numerous circRNAs played a role in the development of AS [7]. For example, circ_0050486 expression was obviously upregulated in AS serum and its silencing might partially abolish oxidized low-density lipoprotein (ox-LDL)-evoked injury in endothelial cells in vitro [8]. Apart from that, the overexpression of circ_0091822 might aggravate ox-LDL-triggered human umbilical vein endothelial cell (HUVEC) damage via regulating cell proliferation, apoptosis, and inflammation [9]. Notably, a recent report suggested that miR-148a-3p might stimulate endothelial cell proliferation and migration, and suppress programmed cell death by the interruption of circ_0003575 (circ_CHMP5) function, thereby exacerbating AS [10]. However, the function of circ_CHMP5 in AS is not completely clear.

CircRNAs are considered to be competitive RNAs of miRNAs, which weakens the effects of miRNAs on their target protein expression [11]. For instance, Liu et al. presented that circIRAK1 might aggravate ox-LDL-induced HUVEC cell apoptosis, inflammatory response, and oxidative stress via absorbing miR-330-5p [12]. Beyond that, Wei et al. exhibited that circ_HECW2 might modulate ox-LDL-triggered cardiovascular endothelial cell dysfunction via targeting the miR-942-5p/TLR4 axis [13]

Ding et al. displayed that ox-LDL enhanced the level of circ_0010283 in vascular smooth muscle cells (VSMCs), and circ_0010283 sponged miR-370-3p to upregulate the expression of HMGB1 to regulate the growth of ox-LDL-treated VSMCs [14]. MiR-516b-5p had been reported to be bound by circAKT3 to inhibit glycolysis in lung cancer cells [15], and miR-516b-5p had also been found to be involved in the development of osteoporosis [16], non-small cell lung cancer (NSCLC) [17], esophageal squamous cell carcinoma [18], etc. Wang et al. disclosed that miR-516b attenuated damage of vascular endothelial cells caused by COCl₂ [19]. But the role of miR-516b-5p is not clear.

As downstream factors of miRNAs, mRNAs are directly involved in biological functions such as protein synthesis [20]. Transforming growth factor beta receptor 2 (TGFβR2) is a type II receptor of TGF-β, the combination of TGFβR2 and TGF-β can regulate cell growth, apoptosis, and other functions [21]. TGFβR2 had been reported to bind to miR-211 and participate in the repair of renal injury [22]. And the proliferation and migration of NSCLC cells were blocked by TGFβR2 insufficiency [23]. TGFβR2 was reported to be negatively regulated by miR-202-5p and took part in the growth of HUVECs [24]. However, the function of TGFβR2 in AS and its relationship with miR-516b-5p and circ_CHMP5 remain unclear.

The study for the first time confirms the role of circ_CHMP5/miR-516b-5p/TGFβR2 axis in ox-LDL-induced HUVECs, and it provides a new potential biomarker for the treatment of AS.

2 Materials and methods

2.1 Patients

23 AS patients and 12 healthy volunteers from Shanxi Provincial People’s Hospital actively provided their blood samples, which were prepared into serum samples for a quantitative real-time polymerase chain reaction (qRT-PCR) experiment. The study was sustained by the Ethics Committee in Shanxi Provincial People’s Hospital [IRB No. (2023) No. 209 of Provincial Medical Sciences].

2.2 Cells

HUVECs were acquired from American Type Culture Collection (Manassas, VA, USA), hatched in Dulbecco’s modified Eagle’s medium (DMEM; Sangon, Shanghai, China) with fetal bovine serum (Sangon) and penicillin-streptomycin (Sangon), and cultured in an incubator containing 5% CO₂ at 37°C. Reaching confluence the cells were passaged with a split ratio of 1 : 3. For the experiments described here HUVECs from passages 3 to 5 were used [25].

AS models were built by treating HUVECs with 100μg/mL of ox-LDL for 24 h.

2.3 QRT-PCR

Total RNA Extraction Kit (TIANGEN, Beijing, China) was obtained to extract RNA, and FastKing One-Step Reverse Transcription-Fluorescent Quantification Kit (SYBR Green) (TIANGEN) was used for qRT-PCR analysis. All primers were shown in Table 1, and the 2^–ΔΔCq method was executed to examine the contents of circ_CHMP5, charged multivesicular body protein 5 (CHMP5), miR-516b-5p, and TGFβR2.

For subcellular localization assay, the RNA in cytoplasm and nucleus was isolated by Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Ontario, Canada), then qRT-PCR was applied to test circ_CHMP5 expression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were reference genes of cytoplasm and nucleus, respectively.

For RNase R assay, 2.5μg RNA dealt with 4 U RNase R (20 U/μL, Abcam) at 37°C for 30 min was the experimental group, and the untreated group was the control group; for actinomycin D assay, 5×10⁴ cells/well was grew in 24-well plates for 24 h and exposed with actinomycin D (2 μg/mL, Abcam) for 0 h, 8 h, 16 h, and 24 h, then the RNA was extracted. The expression levels of circ_CHMP5 and CHMP5 mRNA were calculated by qRT-PCR.

2.4 Cell transfection

Short hairpin RNA (shRNA) against circ_CHMP5 (sh-circ_CHMP5) was constructed to downregulate circ_CHMP5 expression and the control was sh-NC. MiR-516b-5p mimics (miR-516b-5p) (miR-NC was the control) and miR-516b-5p inhibitor (anti-miR-516b-5p) (anti-miR-NC was the control) were used to upregulate or downregulate the expression of miR-516b-5p. The pcDNA was as a control and the TGFβR2 expression vector (TGFβR2) was used to increase TGFβR2 expression. These vectors were got from GenePharma (Shanghai, China) and transfected into 4×10⁵ HUVECs in 96-well plates by means of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The transfection efficiency was measured 24 h after transfection.

2.5 5-Ethynyl-2’-deoxyuridine (EdU) assay

4×10⁴ HUVECs were sowed in 96-well plates and exposed with ox-LDL and sh-circ_CHMP5 respectively for 24 h according to experimental requirements. 100μL EdU (RIBOBIO, Guangzhou, China) was appended into the well and trained for 2 h. Cells were fixed with 50μL cell fixative and 50μL 2 mg/mL glycine, and cleaned with phosphate buffer saline (PBS) (Sangon). Then cells were hatched with 100μL Hoechst33342 reaction solution and scoured with PBS. A Fluorescence microscope was used to observe and take photos.

2.6 Cell counting kit-8 (CCK8) assay

100μL HUVECs were inoculated in 96-well plates and treated with ox-LDL and sh-circ_CHMP5 respectively for 24 h according to experimental requirements. 10μL CCK8 solution (GLPBIO, Montclair, CA, USA) was added to cells at 0 h, 12 h, and 24 h after treatment, and the absorbance at 450 nm was examined by a microplate reader 4 h later.

2.7 Western blot assay

DNA/RNA/ Protein Co-Extraction Kit (TIANGEN) was used to isolate proteins from 2×10⁵ HUVECs in 96-well plates. The appropriate proteins with sample loading buffer were mixed in proper proportion and cooked in boiling water at 100°C for 10 min. Proteins were transferred to the gel block by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific) by wet transfer method. Then, the membrane was sealed by albumin from bovine serum (BSA) (Thermo Fisher Scientific) and cultivated with primary antibodies (anti-Cyclin D1 antibody, ab16663, 1 : 25; anti-proliferating cell nuclear antigen (PCNA) antibody, ab92552, 1 : 1000; anti-B-cell lymphoma-2 (Bcl-2) antibody, ab32124, 1 : 1000; anti-BCL2-Associated X (Bax) antibody, ab32503, 1 : 1000; anti-Cleaved casp-3 antibody, ab2302, 1 : 50; anti-GAPDH antibody, ab8245, 1 : 500; Abcam) and second antibody (ab205718, 1 : 2000, Abcam). Finally, protein bands in membrane were observed under an iBright imaging system using Pro-light HRP chemiluminescence detection reagent (TIANGEN).

2.8 Cell apoptosis assay

Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) was utilized for cell apoptosis. Shortly, 1×10⁴ HUVECs in 96-well plates were reaped and suspended with 195μL binding buffer, 5μL Annexin V-FITC and 10μL propidium iodide (PI) and hatched at 24°C in dark for 20 min, and apoptotic cells were observed by flow cytometry within 1 hour.

2.9 Tube formation assay

10μL Matrigel (BD Biosciences, San Jose, CA, USA) was melted at 4°C and placed in the lower hole of ibidi angiogenesis slide (ibidi, Martin Reid, Germany). After Matrigel gel was solidified, 50μL of treated HUVECs suspension at a concentration of 2×10⁵ cells/mL were appended into the upper pores of the angiogenesis slide, and the cell tube formation was surveyed under a microscope after 12 h of culture.

2.10 Dual-luciferase reporter assay

The wild-type (WT) sequences of circ_CHMP5 or TGFβR2 3’UTR covering the binding sites of miR-516b-5p and their mutated (MUT) sequences were consolidated into the psiCHECK2 vectors (Promega, Madison, WI, USA), naming circ_CHMP5-WT, circ_CHMP5-MUT, TGFβR2-WT and TGFβR2-MUT. They were transfected into 2×10⁴ HUVECs in 96-well plates with miR-516b-5p or miR-NC, and the luciferase activity was assessed with the help of Dual-Luciferase® Reporter Assay System (Promega).

2.11 RNA-pull down assay

Biotin-labeled circ_CHMP5-WT (Bio-circ_CHMP5-WT), Bio-NC, Bio-circ_CHMP5-MUT, Bio-TGFβR2-WT and Bio-TGFβR2-MUT were built by Sangon (Shanghai, China) and hatched with HUVECs. Then, 1×10⁵ HUVECs in 96-well plates were collected after 48 h and trained with magnetic beads. Then miR-516b-5p expression was tested via qRT-PCR.

2.12 Statistical analysis

All cellular experiments were independently repeated three times. The data in this study were analyzed using SPSS 22.0 (IBM, Chicago, IL, USA) and GraphPad Prism 8.0 software (GraphPad Inc., LaJolla, California, USA). The difference between two sets of data was estimated by Student’s t-test, and the significance of more than three groups of data was computed using one-way analysis of variance (ANOVA). Pearson’s correlation analysis was used to evaluate the relationship between miR-516b-5p and circ_CHMP5 or TGFβR2. The final results were demonstrated by mean±standard deviation. P < 0.05 was considered to be statistically significant.

3 Results

3.1 Circ_CHMP5 was upregulated in AS patients and ox-LDL-treated HUVECs

As illustrated in Fig.1A, circ_CHMP5 was derived from exons 5, 6, and 7 of the CHMP5 gene. We found circ_CHMP5 was significantly promoted in the serum of patients with AS (Fig.1B), and was dramatically facilitated in ox-LDL-treated HUVECs (Fig.1 C). The results of subcellular localization showed that circ_CHMP5 mainly existed in the cytoplasm (Fig.1D). After RNA treated with RNase R, there was no significant change in circ_CHMP5 expression, while the mRNA expression of linear CHMP5 was obviously decreased, suggesting that circ_CHMP5 was resistant to RNase R (Fig.1E). The data of actinomycin D assay showed that the circ_CHMP5 half-life was longer than that in CHMP5 mRNA (Fig.1F). In a word, circ_CHMP5 mainly existed in the cytoplasm, and was boosted in serum of AS patients.

Fig. 1

Circ_CHMP5 was upregulated in AS patients and ox-LDL-treated HUVECs. (A) Schematic diagram of the genomic location and characteristics of circ-CHMP5 (294 bp). (B and C) QRT-PCR assay for the expression of circ_CHMP5 in serum of AS patients and ox-LDL treated HUVECs. (D) Subcellular localization assay was used to detect the position of circ_CHMP5 in HUVECs. (E and F) RNase R and actinomycin D assays were used to identify the circular structure of circ_CHMP5. *P < 0.05.

3.2 Circ_CHMP5 knockdown promoted the growth of ox-LDL-treated HUVECs

The inhibition efficiency of sh-circ_CHMP5 transfection was detected, and the data showed that the content of circ_CHMP5 was notably blocked by sh-circ_CHMP5 introduction, while the expression of CHMP5 mRNA was not changed (Fig.2A). A markedly inhibition of cell proliferation was observed in ox-LDL-treated HUVECs, while circ_CHMP5 deficiency partly reversed the effect of ox-LDL on proliferation (Fig.2B). The CCK8 assay indicated that after transfected 24 h, silence of circ_CHMP5 overturned the inhibitory effect of ox-LDL on cell viability in HUVECs (Fig.2 C). The ox-LDL suppressed the expression of cycle factor Cyclin D1 and proliferating factor PCNA in HUVECs, and the suppressive effects were abolished after sh-circ_CHMP5 was transfected (Fig.2D). Downregulation of circ_CHMP5 attenuated the pro-apoptotic effect of ox-LDL exposure on HUVECs (Fig.2E). In ox-LDL-stimulated HUVECs, the expression of anti-apoptotic factor Bcl-2 was remarkably reduced, while the expression levels of apoptotic promoter Bax and Cleaved casp-3 were increased, and these effects were overturned after transfection with sh-circ_CHMP5 (Fig.2F). The capacity of tube formation in ox-LDL+sh-circ_CHMP5 group was strikingly elevated than that in ox-LDL group (Fig.2 G). In short, circ_CHMP5 knockdown reinforced the proliferation, viability and tube formation, and confined cell apoptosis of ox-LDL-treated HUVECs.

Fig. 2

Circ_CHMP5 knockdown regulated the growth of ox-LDL-stimulated HUVECs. (A) QRT-PCR was carried to test the transfected efficiency of sh-circ_CHMP5. (B) EdU assay was performed to detect cell proliferation. (C) Cell viability was tested by CCK8 assay. (D) Western blot assay was applied for proteins expression. (E) Flow cytometry was used to measure cell apoptosis. (F) The protein expression of Bcl-2, Bax and Cleaved casp-3 in ox-LDL-treated HUVECs were measured by western blot assay. (G) Tube formation assay was used to detect tube formation capacity. *P < 0.05.

3.3 MiR-516b-5p was regulated by circ_CHMP5

The binding sites between miR-516b-5p and circ_CHMP5 were predicted by circinteractome (https://circinteractome.nia.nih.gov/) (Fig.3A). The luciferase activity of circ_CHMP5-WT was greatly inhibited by miR-516b-5p and the enrichment of miR-516b-5p was increased by Bio-circ_CHMP5-WT, indicating that miR-516b-5p could directly bind to circ_CHMP5 (Fig.3B-C). And the level of miR-516b-5p was expedited by sh-circ_CHMP5 addition (Fig.3D). We also found that miR-516b-5p was downregulated in the serum of AS patients, and the expression of miR-516b-5p was negatively correlated with the expression of circ_CHMP5 (Fig.3E-F). And ox-LDL inhibited the level of miR-516b-5p in HUVECs (Fig.3 G). Collectively, miR-516b-5p was hampered in AS patients and ox-LDL-exposure HUVECs, and was a target of circ_CHMP5.

Fig. 3

Circ_CHMP5 functioned as a sponge of miR-516b-5p. (A) The binding sites between miR-516b-5p and circ_CHMP5 were predicted by circinteractome (https://circinteractome.nia.nih.gov/). (B) Luciferase activities of vectors were detected through dual-luciferase reporter assay. (C) RNA-pull down assay was used to detect the enrichment of miR-516b-5p. (D and E) QRT-PCR was utilized for the expression of miR-516b-5p in HUVECs and in serum of AS patients. (F) Pearson’s correlation analysis determined the association between miR-516b-5p expression and circ_CHMP5 expression in AS patients. (G) The expression of miR-516b-5p in HUVECs was estimated by QRT-PCR. *P < 0.05.

3.4 The effects of circ_CHMP5 depletion on ox-LDL-exposed HUVECs were abated by miR-516b-5p decrease

Anti-miR-516b-5p transfection relieved the promotion effect of circ_CHMP5 knockdown on miR-516b-5p expression (Fig.4A). In ox-LDL-exposed HUVECs, miR-516b-5p insufficiency weakened the elevation effects of circ_CHMP5 knockdown on proliferation, viability, Cyclin D1 and PCNA (Fig.4B-D). Downregulation of circ_CHMP5 repressed cell apoptosis in ox-LDL-stimulated HUVECs, and the influence was regained after anti-miR-516b-5p co-transfection (Fig.4E). Lack of miR-516b-5p ameliorated the effects of silencing circ_CHMP5 on Bcl-2, Bax and Cleaved casp-3 in ox-LDL-exposed HUVECs (Fig.4F). The tube formation assay disclosed that knockdown of circ-_CHMP5 promoted the tube formation, and the effect was restored in ox-LDL-treated HUVECs co-transfected with anti-miR-516b-5p (Fig.4 G). In brief, circ_CHMP5 regulated the growth of ox-LDL-treated HUVECs through miR-516b-5p.

Fig. 4

Circ_CHMP5 regulated the progression of HUVECs though miR-516b-5p. (A) MiR-516b-5p expression was examined by qRT-PCR in HUVECs transfected with sh-NC, sh-circ_CHMP5, sh-circ_CHMP5 + anti-miR-NC, sh-circ_CHMP5 + anti-miR-516b-5p. (B-G) HUVECs were treated with ox-LDL, sh-circ_CHMP5 or sh-circ_CHMP5 + anti-miR-516b-5p as well as the homologous controls. (B and C) The proliferation and viability were tested by EdU and CCK8 assays, respectively. (D) The protein expression levels of Cyclin D1 and PCNA were determined by western blot. (E) The cell apoptosis of HUVECs was tested by flow cytometry. (F) Western blot was performed to test the protein levels of Bcl-2, Bax and Cleaved casp-3. (G) The tube formation capacity was detected by tube formation assay. *P < 0.05.

3.5 TGFβR2 was the target of miR-516b-5p

Starbase (https://starbase.sysu.edu.cn/) was used to forecast the target of miR-516b-5p, and the combination sequence between miR-516b-5p and TGFβR2 was displayed in Fig.5A. The results of dual-luciferase reporter assay and RNA-pull down assay disclosed that miR-516b-5p bound TGFβR2 in HUVECs (Fig.5B-C). The transfected efficiencies of miR-516b-5p and anti-miR-516b-5p were tested, and the data implied that miR-561b-5p was reinforced by miR-516b-5p overexpression, and curbed by miR-516b-5p downregulation (Fig.5D). And TGFβR2 was negatively regulated by miR-516b-5p (Fig.5E-F). In addition, TGFβR2 was specially boosted in serum of AS patients and the expression of TGFβR2 was passively correlated with the expression of miR-516b-5p, and was positively correlated with circ_CHMP5 expression (Fig.5G-J). The mRNA and protein expression levels of TGFβR2 were robustly intensified in HUVECs by ox-LDL (Fig.5K-L). And in HUVECs, circ_CHMP5 knockdown prominently retarded the expression of TGFβR2, and anti-miR-516b-5p co-transfection abrogated the effect (Fig.5M-N). The data displayed that TGFβR2 was regulated by miR-516b-5p.

Fig. 5

TGFβR2 was the downstream gene of miR-516b-5p. (A) The binding sequence between miR-516b-5p and TGFβR2. (B and C) The relationship between miR-516b-5p and TGFβR2 were confirmed by dual-luciferase reporter assay and RNA-pull down assay. (D) The transfected efficiencies of miR-516b-5p and anti-miR-516b-5p were measured by qRT-PCR. (E and F) The mRNA and protein expression of TGFβR2 in HUVECs transfected with miR-516b-5p or anti-miR-516b-5p were determined by qRT-PCR and western blot. (G and H) The level of TGFβR2 in serum of AS patients was measured by qRT-PCR and western blot. (I and J) The associations between TGFβR2 expression and miR-516b-5p expression or circ_CHMP5 expression in patients with AS were analyzed by Pearson’s correlation analysis. (K and L) QRT-PCR and western blot were performed to detect the effect of ox-LDL on TGFβR2 in HUVECs. (M and N) After HUVECs transfected with sh-NC, sh-circ_CHMP5, sh-circ_CHMP5 + anti-miR-NC or sh-circ_CHMP5 + anti-miR-516b-5p, the content of TGFβR2 was tested by qRT-PCR and western blot. *P < 0.05.

3.6 MiR-516b-5p regulated the growth of ox-LDL-treated HUVECs though TGFβR2

The repressive effect of miR-516b-5p on TGFβR2 was reverted by TGFβR2 overexpression (Fig.6A-B). As shown in Fig.6C-D, cell proliferation and viability were obviously fortified in ox-LDL-exposure HUVECs by miR-516b-5p overexpression and TGFβR2 addition partly overturned these effects. And overexpression of miR-516b-5p greatly reinforced the protein expression levels of Cyclin D1 and PCNA in ox-LDL-treated HUVECs, and these impacts were overturned by TGFβR2 increase (Fig.6E). TGFβR2 overexpression neutralized the suppressive effect of miR-516b-5p on cell apoptosis in ox-LDL-induced HUVECs (Fig.6F). The effects of miR-516b-5p on Bcl-2, Bax, Cleaved casp-3 and tube formation in ox-LDL-stimulated HUVECs were abolished by TGFβR2 replenishing (Fig.6G-H). The above data disclosed that TGFβR2 upregulation recuperated the effects of miR-516b-5p on ox-LDL-induced HUVECs.

Fig. 6

MiR-516b-5p regulated the process of ox-LDL-treated HUVECs by TGFβR2. (A and B) QRT-PCR and western blot were used to determine the expression of TGFβR2 after HUVECs treated with miR-NC, miR-516b-5p, miR-516b-5p+pcDNA or TGFβR2. (C-H) HUVECs were treated with 0 or 100μg/mL of ox-LDL, ox-LDL+miR-nc, ox-LDL+miR-516b-5p, ox-LDL+miR-516b-5p+pcDNA or ox-LDL+miR-516b-5p+TGFβR2. (C and D) EdU and CCK8 assays were used to test cell proliferation and viability. (E) Western blot was utilized to detect the protein expression of Cyclin D1 and PCNA. (F) Flow cytometry was performed to measure cell apoptosis. (G) The contents of Bcl-2, Bax and Cleaved casp-3 were determined by western blot. (H) Tube formation capacity was detected by tube formation assay. *P < 0.05.

4 Discussion

Atherosclerosis, which is the narrowing of the inside of arteries due to the buildup of plaque [26] and begins in early adulthood and increases with age and a sedentary lifestyle [27]. As a vascular disease with high mortality [28], the cure for atherosclerosis has always been a challenge for medical workers.

Genetic factors are important causes of cancers, and circRNAs have been proven to play a vital role in the occurrence and development of cancers. For instance, circ_0075960 targeted miR-361-3p to regulate SH2B1 expression to regulate the progression of endometrial carcinoma [29]. Shang et al. disclosed that miR-148a-3p hindered the expression circ_0003575 to elevate cell viability and migration of endothelial cells [10]. We demonstrated that circ_CHMP5 was boosted in AS patients and ox-LDL-exposure HUVECs, and circ_CHMP5 silencing attenuated the effects of ox-LDL on proliferation, tube formation, and apoptosis in HUVECs. Similarly, Li et al. manifested that circ_0003575 was increased in ox-LDL-treated HUVECs, and the proliferation and tube formation capacity of ox-LDL-exposure HUVECs were reinforced by circ_0003575 downregulation [30]. At the same time, we found that the protein levels of Cyclin D1, PCNA, and Bcl-2 in ox-LDL-treated HUVECs were enhanced by circ_CHMP5 knockdown, and silencing circ_CNMP5 limited the expression of Bax and Cleaved casp-3. All data indicated that circ_CHMP5 reversed the damage of ox-LDL on HUVECs.

Many studies have shown that circRNAs could function as a sponge for miRNAs, and circBank, circBase, and circinteractome could be used to screen circRNAs targets. We revealed that miR-516b-5p might be the target of circ_CHMP5 by using circinteractome, which was ensured by dual-luciferase reporter assay and RNA-pull down assay. There are few studies on miR-516b-5p, mainly focusing on bone [31] and fetal growth [32, 33]. MiR-516b-5p had been shown to inhibit the growth of bladder cancer cells [34]. Besides, miR-516b-5p intensified viability, tube formation and migration of vascular endothelial cells [35]. Our present study revealed that miR-516b-5p was restrained in AS patients and ox-LDL-treated HUVECs and was negatively regulated by circ_CHMP5. Furthermore, miR-516-5p expedited the proliferation and tube formation of ox-LDL-stimulated HUVECs and hampered cell apoptosis. Moreover, the effects of sh-circ_CHMP5 on ox-LDL-triggered HUVECs were overturned by miR-516b-5p lack. The results of our study speculated that circ_CHMP5 fortified the progression of ox-LDL-treated HUVECs through miR-516b-5p.

Here, TGFβR2 was found to be the downstream gene of miR-516b-5p and was co-regulated by miR-516b-5p and circ_CHMP5. TGFβR2 was reported to be a tumor repressor gene in colorectal cancer [36], and TGFβR2 was associated with the apoptosis of goat granulosa cells [37]. LncRNA NEAT1 regulated the procedure of gastric cancer via miR-17-5p and TGFβR2 [38]. In our study, TGFβR2 was obviously impelled in serum of AS patients, and ox-LDL aggrandized the expression of TGFβR2 in HUVECs. Moreover, overexpression of TGFβR2 relieved the effects of miR-516b-5p in ox-LDL-exposure HUVECs. Gu et al. also demonstrated that TGFβR2 was involved in the proliferation, tube formation, and migration of HUVECs [24]. In a word, after HUVECs treated with ox-LDL, circ_CHMP5 inhibited TGFβR2 expression through miR-516b-5p, aggravated the injury of ox-LDL to HUVECs, inhibited cell proliferation and tube formation, and promoted apoptosis (Fig.7).

Fig. 7

The schematic diagram of circ_CHMP5/miR-516b-5p/TGFβR2 axis working in HUVECs tumorigenesis.

5 Conclusion

We explored a new pathway of circ_CHMP5 regulation in HUVECs: circ_CHMP5 regulated the occurrence and development of ox-LDL-revulsive HUVECs by miR-516b-5p/TGFβR2 axis. The results provided a new biological target for the prognosis and treatment of AS.

Footnotes

Acknowledgments

None.

Disclosure of interest

The authors declare that they have no conflicts of interest.

Funding

None.

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