Knockdown of circ_0113656 assuages oxidized low-density lipoprotein-induced vascular smooth muscle cell injury through the miR-188-3p/IGF2 pathway

2.1 Study subjects

Blood samples were collected from AS patients (N = 17) and healthy volunteers (N = 13) at the People’s Hospital of Jiangxi Provincial. Plaque sites and ranges were evaluated using a Madison ultrasound system. Besides, two neurologists identified the diseases. AS patients with other clinical diseases were excluded. Blood samples were centrifuged at 1,000g for 10 min and then stored in a refrigerator.

1. Ethical approval: The research related to human use has complied with all the relevant national regulations and institutional policies, in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of People’s Hospital of Jiangxi Provincial. All participants signed the written informed consent.

2.2 Cell culture and ox-LDL stimulation

HVSMCs were purchased from Tongpai Biotech (Shanghai, China) and maintained in Ham’s F12K medium (Sunncell, Wuhan, China) plus 10% fetal bovine serum (Sunncell) and 1% penicillin/streptomycin (Sunncell) at 37°C in an incubator buffered with 5% CO₂. To disclose the molecular mechanism behind circ_0113656 regulating HVSMC injury, HVSMCs were stimulated using ox-LDL (Yeasen Biotech, Shanghai, China; 0, 25, 50, and 100 µg/mL) for 24 h as shown previously [17].

2.3 Cell transfection

Small interfering RNA of circ_0113656 (si-circ_0113656, 5′-GGCAGAGCCCAGCAAGATTGT-3′), miR-188-3p mimics (miR-188-3p, 5′-CUCCCACAUGCAGGGUUUGCA-3′), miR-188-3p inhibitors (anti-miR-188-3p, 5′-UGCAAACCCUGCAUGUGGGAG-3′) and the corresponding negative controls (si-NC, miR-NC, and anti-miR-NC) were synthesized by Ribobio Co., Ltd. (Guangzhou, China). The full-length coding sequence of IGF2 was amplified and inserted into the pcDNA 3.1(+) vector to generate the overexpression plasmid of IGF2. In strict accordance with the guidebook of Lipofectamine 3000, transfection was performed on HVSMCs maintained in 12-well plates in an atmosphere containing 5% CO₂. Transfection efficiency was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).

2.4 Cell viability

HVSMCs transfected with plasmids and oligonucleotides or treated with ox-LDL were maintained in 96-well plates for 48 h. Then, the cell counting kit-8 (CCK-8) reagent (Dojindo, Shanghai, China) was used to incubate the cells according to the manufacturer’s direction. Finally, these samples were analyzed using an enzyme immunoassay analyzer (Azure Ao; Cycloud Biotech, Beijing, China).

2.5 Cell proliferation analysis

The assay was performed to determine the proliferative ability of HVSMCs with various treatments. In brief, HVSMCs were treated and allowed to grow in 12-well plates for 48 h. The cells were subcultured in 96-well plates supplemented with EdU-labeled Ham’s F12K (Sunncell) for 2 h and then stained using 4′,6-diamidino-2-phenylindole (Sbjbio® life science, Nanjing, China). At last, the stained cells from five random fields were analyzed using a confocal microscope (Olympus, Tokyo, Japan).

2.6 Transwell invasion assay

Transwell analysis involving the use of transwell compartments with Matrigel (Corning, Beijing, China) was performed to evaluate the invasive capacity of HVSMCs. In brief, cells with various treatments were allowed to culture in the upper chambers, which were pre-added with serum-free Ham’s F12K medium (Sunncell), whereas the lower chambers were supplemented with Ham’s F12K medium plus 15% serum. After 24 h incubation, the cells invaded into the lower chambers and were dyed using crystal violet (Yaji Biotech, Shanghai, China) and then counted under an inverted microscope (100× magnification; Olympus).

2.7 Wound-healing assay

HVSMCs at 80–90% confluence were treated with plasmids, oligonucleotide, and ox-LDL alone or jointly. After 14 days of culture, sterile 10 μL tips were applied to scratch the cells, and then the cells went through 24 h culture. The images of wounds were captured at 0 and 24 h using an inverted microscope (Olympus).

2.8 Western blotting analysis

Total proteins from the blood samples of AS patients and HVSMCs were isolated using RIPA lysis buffer containing a protease inhibitor (Sbjbio^® life science). The concentrations of extracted proteins were determined with a BCA protein assay kit (Sbjbio^® life science). Then, polyacrylamide gels were used to separate protein with a SureLock Midi-Cell Electrophoresis System. After blocking the aspecific signals using skimmed milk (Yili, Beijing, China), polyvinylidene fluoride membranes with protein bands were incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA) (#13-3900; 1:5000; Thermo Fisher, Waltham, MA, USA), matrix metalloprotein 2 (MMP2) (#436000; 1:250; Thermo Fisher), IGF2 (#MA5-17096; 1:1,000; Thermo Fisher), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#MA1-16757; 1:2,000; Thermo Fisher). After being exposed to eyoECL Plus (Beyotime, Shanghai, China), the visualized blots were analyzed using Image J software.

2.9 qRT-PCR and RNA treatment

TRIzol™ reagent (#15596026; Thermo Fisher) was used to prepare cell lysates as per the guidebook. A NanoDrop spectrophotometer was applied to detect RNA concentration. cDNA synthesis reagents (Vazyme, Jiangsu, China) and miRNA first strand synthesis kit (Vazyme) were used for reverse transcription. Then, diluted cDNA was subjected to qRT-PCR analysis on a qRT-PCR system (Bio-Rad, Hercules, CA, USA) with SYBR qPCR Master Mix (Vazyme). The expression of circ_0113656, miR-188-3p, and IFG2 was normalized to GAPDH or U6 by the 2^−∆∆Ct method. Random primers and oligo(dT)₁₈ primers were utilized to identify the circular structure of circ_0113656. Besides, 1 μg of RNA was exposed to RNase R (Xiyuan Biotech, Shanghai, China) at 37°C for 20 min to analyze the circRNA structure. Primers used for amplification are listed in Table 1.

2.10 RNA immunoprecipitation (RIP) assay

The assay was performed based on the guidebook of a Magna RIP kit (Sigma, St. Louis, MO, USA). In brief, 1 × 10⁵ HVSMCs cultured in 12-well plates were collected and lysed using RIP lysis buffer (Sbjbio^® life science). Then, the lysates were incubated with anti-Ago2-conjugated magnetic beads for 24 h with an IgG antibody as a negative control. Finally, the expression of circ_0113656, miR-188-3p, and IGF2 in the complexes enriched with magnetic beads was quantified by qRT-PCR.

2.11 Dual-luciferase reporter assay

The binding sites of miR-188-3p for circ_0113656 and IFG2 were predicted using online databases including Circinteractome (https://circinteractome.nia.nih.gov/index.html), Circbank (http://www.circbank.cn/index.html), and Targetscan (http://www.targetscan.org/vert_71/). The wild-type (WT) reporter plasmids including WT-circ_0113656 and WT-IGF2 3′-untranslated region (3′-UTR) were generated by introducing the sequences of circ_0113656 and IGF2 3′-UTR with the binding sites of miR-188-3p into the pmirGLO vector. The mutant plasmids including MUT-circ_0113656 and MUT-IGF2 3′-UTR were built using site mutagenesis kits (Yeasen Biotech). Then, the reporter plasmids together with miR-188-3p mimics or mimic control were transfected into HVSMCs for 48 h. At last, the dual-Lucy assay kit (Solarbio, Beijing, China) was utilized to analyze the binding intensity.

2.12 Statistical analysis

All data from three independent duplicate tests were analyzed by GraphPad Prism and presented as mean ± standard deviations (SD). The significant differences were compared with Mann–Whitney U test, Student’s t-test, or analysis of variance. Spearman correlation analysis was used to analyze the interactions of miR-188-3p expression with circ_0113656 and IGF2 expression. P < 0.05 indicated a statistical significance.

3.1 Ox-LDL treatment induces HVSMC injury

HVSMCs cells were treated with ox-LDL at various concentrations (0, 25, 50, and 100 µg/mL) for 24 h to mimic AS-like injury in vivo and explore the consequential effects on cell proliferation, invasion, and migration. As shown in Figure 1a and b, ox-LDL treatment dose-dependently promoted HVSMC viability and proliferation. Consistently, the invasion and migration of HVSMCs were increased after ox-LDL treatment in a dose-dependent manner (Figure 1c–e). As expected, the expression of the two proteins was dose-dependently upregulated by ox-LDL treatment (Figure 1f). These results demonstrate that ox-LDL treatment indeed induces HVSMC injury. Based on the above results, HVSMCs were treated with 50 µg/mL ox-LDL for 24 h in the following study.

Figure 1

Ox-LDL induces HVSMC injury. HVSMCs were treated with various concentrations of ox-LDL (0, 25, 50, and 100 µg/mL), and cell viability was analyzed by CCK-8 (a), cell proliferation by EdU assay (b), cell invasion by transwell assay (c), cell migration by wound-healing assay (d and e), and the protein expression of PCNA and MMP2 by Western blotting analysis (f). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.2 Circ_0113656 knockdown assuages ox-LDL-induced HVSMC disorders

Then, we detected circ_0113656 expression in the blood samples of AS patients. As shown in Figure 2a, circ_0113656 expression was significantly increased in the blood of AS patients in comparison with healthy controls. Moreover, circ_0113656 expression was increased in ox-LDL-stimulated HVSMCs in a concentration-dependent manner (Figure 2b). Subsequently, our results identified the circular structure of circ_0113656 using RNase R, random primers, and oligo(dT)₁₈ primers. For instance, RNase R treatment greatly reduced GAPDH expression but it had no significant effect on circ_0113656 expression (Figure 2c). Meanwhile, PCR production of circ_0113656 amplified using random primers was more than that amplified using oligo(dT)₁₈ primers (Figure 2d). Based on the above results, we silenced circ_0113656 in ox-LDL-treated HVSMCs to analyze the consequent effects on cell proliferation, invasion, and migration. The results of qRT-PCR showed that ox-LDL treatment increased circ_0113656 expression, which was attenuated after circ_0113656 depletion (Figure 2e). Comparatively, ox-LDL-induced promotion in cell viability, proliferation, invasion, and migration were relieved after circ_0113656 silencing (Figure 2f–i). Besides, the increased expression of PCNA and MMP2 by ox-LDL was remitted when circ_0113656 expression was downregulated (Figure 2j).

Figure 2

The effects of circ_0113656 on ox-LDL-induced HVSMC injury. (a) Circ_0113656 expression was detected by qRT-PCR in the blood samples of AS patients and healthy volunteers (N = 17 and N = 13, respectively). (b) Circ_0113656 expression was detected by qRT-PCR in HVSMCs treated with ox-LDL (0, 25, 50, and 100 µg/mL). (c and d) The circular structure of circ_0113656 was identified by using RNase R, random primers, and oligo(dT)₁₈ primers. HVSMCs were divided into the ox-LDL group, ox-LDL + si-NC group, and ox-LDL + si-circ_0113656 group, with untreated HVSMCs as controls, and circ_0113656 expression was analyzed by qRT-PCR (e), cell viability by CCK-8 (f), cell proliferation by EdU assay (g), cell invasion by transwell assay (h), cell migration by wound-healing assay (i), and the protein expression of PCNA and MMP2 by Western blotting analysis (j). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.3 Circ_0113656 acts as a miR-188-3p sponge

Circular RNA interactome (Circinteractome) and circBank online databases were used to predict the target miRNAs of circ_0113656. As presented in Figure 3a, we found four miRNAs that contained the complementary sites of circ_0113656, including miR-149-5p, miR-184, miR-602, and miR-188-3p, by overlapping the prediction results of the two online databases. Given higher miR-188-3p expression in HVSMCs transfected with si-circ_0113656 (Figure 3a), the miRNA was employed as a follow-up subject. The binding sites of circ_0113656 for miR-188-3p are shown in Figure 3b. Subsequently, we identified the regulatory relationship of circ_0113656 for miR-188-3p using dual-luciferase reporter assay and RIP assay. The success of miR-188-3p overexpression is presented in Figure 3c. As shown in Figure 3d, miR-188-3p introduction significantly inhibited the luciferase activity of wt-circ_0113656 but not that of mut-circ_0113656. Also, miR-188-3p and circ_0113656 could be greatly enriched in the Ago2 antibody group in comparison with the IgG antibody group (Figure 3e). We observed that miR-188-3p expression was significantly downregulated and was negatively correlated with circ_0113656 expression in the blood samples of AS patients (Figure 3f and g). Further, ox-LDL treatment reduced miR-188-3p expression in HVSMCs (Figure 3h) in a concentration-dependent manner.

Figure 3

MiR-188-3p targets circ_0113656 in HVSMCs. (a) The miRNAs containing the complementary sites of circ_0113656 were predicted by Circinteractome and circBank online databases. (b) Schematic illustration showing the binding sites of circ_0113656 for miR-188-3p. (c) The efficiency of miR-188-3p mimics in increasing miR-188-3p expression was detected by qRT-PCR in HVSMCs. (d and e) Dual-luciferase reporter assay and RIP assay were carried out to determine the association between miR-188-3p and circ_0113656. (f) MiR-188-3p expression was detected by qRT-PCR in the blood samples of AS patients and healthy volunteers (N = 17 and N = 13, respectively). (g) Spearman correlation analysis was used to determine the linear correlation between miR-188-3p and circ_0113656 expression in the blood samples of AS patients. (h) MiR-188-3p expression was checked by qRT-PCR in HVSMCs treated with ox-LDL (0, 25, 50, and 100 µg/mL). *P < 0.05, ***P < 0.001, and ****P < 0.0001.

3.4 Circ_0113656 regulates ox-LDL-induced HVSMC disorders by binding to miR-188-3p

We silenced circ_0113656 and miR-188-3p to analyze the consequential effects in ox-LDL-induced HVSMCs. As presented in Figure 4a, circ_0113656 knockdown-induced upregulation of miR-188-3p was attenuated after miR-188-3p depletion in ox-LDL-treated HVSMCs. Subsequently, we observed that circ_0113656 depletion-induced inhibition in cell viability, proliferation, invasion, and migration were remitted when miR-188-3p expression was downregulated in ox-LDL-treated HVSMCs (Figure 4b–f). Circ_0113656 knockdown significantly reduced PCNA and MMP2 expression in ox-LDL-treated HVSMCs, whereas these effects were rescued after transfection with miR-188-3p inhibitors (Figure 4g).

Figure 4

The effects of circ_0113656 knockdown and miR-188-3p depletion on ox-LDL-induced HVSMC injury. HVSMCs were divided into the ox-LDL group, ox-LDL + si-NC group, ox-LDL + si-circ_0113656 group, ox-LDL + si-circ_0113656 + anti-miR-NC group, and ox-LDL + si-circ_0113656 + anti-miR-188-3p group, with untreated HVSMCs as controls. MiR-188-3p expression was analyzed by qRT-PCR (a), cell viability by CCK-8 (b), cell proliferation by EdU assay (c), cell invasion by transwell assay (d and e), cell migration by wound-healing assay (f), and the protein expression of PCNA and MMP2 by Western blotting analysis (g). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.5 MiR-188-3p binds to IGF2 in HVSMCs

As predicted by Targetscan online database, IGF2 contained the binding sites of miR-188-3p (Figure 5a). We then performed dual-luciferase reporter assay and RIP assay to determine whether miR-188-3p targeted IGF2. As expected, the luciferase activity of WT-IGF2 3′-UTR was significantly reduced after transfection with miR-188-3p mimics, whereas that of MUT-IGF2 3′-UTR had no response to miR-188-3p introduction (Figure 5b). Meanwhile, miR-188-3p and IGF2 expression in the co-precipitated RNAs induced by the Ago2 antibody were higher than their expression in the co-precipitated RNAs induced by the IgG antibody (Figure 5c). These data suggest that miR-188-3p targets IGF2. Consistently, we found that IGF2 expression was greatly increased and negatively correlated with miR-188-3p expression in the blood samples of AS patients (Figure 5d–f). Further, ox-LDL treatment promoted IGF2 production in a concentration-dependent manner (Figure 5g).

Figure 5

IGF2 is identified as a target of miR-188-3p. (a) Schematic illustration showing the complementary sites of miR-188-3p with IGF2. (b and c) Dual-luciferase reporter assay and RIP assay were performed to analyze the association between miR-188-3p and IGF2. (d and f) The mRNA and protein expression of IGF2 was detected by qRT-PCR and Western blotting analysis, respectively, in the blood samples from AS patients and healthy volunteers. (e) Spearman correlation analysis was used to determine the linear correlation between miR-188-3p and IGF2 expression in the blood samples of AS patients. (g) IGF2 protein expression was checked by Western blotting analysis in HVSMCs treated with ox-LDL (0, 25, 50, and 100 µg/mL). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.6 IGF2 overexpression remits miR-188-3p-mediated effects in ox-LDL-treated HVSMCs

Given the association between miR-188-3p and IGF2, we further analyzed whether IGF2 participated in miR-188-3p-induced effects in ox-LDL-treated HVSMCs. The study first showed that miR-188-3p mimics reduced IGF2 expression in ox-LDL-treated HVSMCs, whereas the effect was attenuated after IGF2 overexpression (Figure 6a). Subsequently, miR-188-3p introduction inhibited cell viability, proliferation, invasion, and migration; however, these effects were remitted when IGF2 was overexpressed (Figure 6b–f). In addition, miR-188-3p-induced inhibition in PCNA and MMP2 expression was restored by the enforced IGF2 expression (Figure 6g). Moreover, we analyzed the effect of IGF2 overexpression on the change of HVSMC phenotypes. The efficiency of IGF2 overexpression is shown in Figure A1a. Then, the data showed that IGF2 overexpression promoted HVSMC viability, proliferation, invasion, migration, and the protein expression of PCNA and MMP2 (Figure A1b–f).

Figure 6

The effects of miR-188-3p and IGF2 on ox-LDL-induced HVSMC disorders. HVSMCs were divided into the ox-LDL group, ox-LDL + miR-NC group, ox-LDL + miR-188-3p group, ox-LDL + miR-188-3p + pcDNA group, and ox-LDL + miR-188-3p + IGF2 group, with mock HVSMCs as controls. IGF2 expression was analyzed by Western blotting analysis (a), cell viability by CCK-8 (b), cell proliferation by EdU assay (c), cell invasion by transwell assay (d and e), cell migration by wound-healing assay (f), and the protein expression of PCNA and MMP2 by Western blotting analysis (g). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3.7 Circ_0113656 depletion reduces IGF2 expression through miR-188-3p in ox-LDL-treated HVSMCs

Based on the above findings, we continued to explore whether IGF2 was the downstream gene of the circ_0113656/miR-188-3p axis in ox-LDL-treated HVSMCs. We silenced circ_0113656 and miR-188-3p in the cells and then detected IGF2 expression using qRT-PCR and Western blotting analysis. As presented in Figure 7a and b, circ_0113656 knockdown significantly reduced the mRNA and protein expression of IGF2 with ox-LDL treatment; however, these effects were remitted when miR-188–3p expression was downregulated. These results demonstrate that circ_0113656 regulates IGF2 expression through miR-188-3p.

Figure 7

The effects of circ_0113656 knockdown and miR-188-3p depletion on IGF2 expression in ox-LDL-treated HVSMCs. HVSMCs were divided into the ox-LDL group, ox-LDL + si-NC group, ox-LDL + si-circ_0113656 group, ox-LDL + si-circ_0113656 + anti-miR-NC group, and ox-LDL + si-circ_0113656 + anti-miR-188-3p group, with untreated HVSMCs as controls. IGF2 mRNA expression was detected by qRT-PCR (a) and IGF2 protein expression by Western blotting analysis (b). **P < 0.01, ***P < 0.001, and ****P < 0.0001.