Background
Many types of cells are involved in the pathogenetic process of atherosclerosis (AS), containing vascular endothelial cells and smooth muscle cells [
1]. Endothelial injury theory is one of the mainstream theories of atherosclerosis pathogenesis, and it considers artery atheromatous plaque as the product of endothelial injury [
2]. Oxidized low-density lipoprotein (ox-LDL) is a crucial risk factor that is responsible for AS initiation [
3]. Therefore, we established AS cell model using ox-LDL-treated HUVECs to find the pivotal molecules that were involved in AS pathogenesis in vitro.
Circular RNAs (circRNAs) are endogenous RNAs without 5′ or 3′ polarity [
4,
5]. CircRNAs have been demonstrated to modulate the pathological process of human malignancies [
6,
7]. Also, accumulating studies have identified the important roles of circRNAs in AS. For instance, Li et al. found that circ_0003575 knockdown accelerated the proliferation ability and tube formation capacity of HUVECs [
8]. Liu et al. demonstrated that circ_0003204 suppressed proliferation and angiogenesis of ox-LDL-treated HUVECs [
9]. Circ_0004104 was reported to be notably up-regulated in patients diagnosed with coronary artery disease [
10]. However, the role and mechanism of circ_0004104 in AS progression remain largely unknown.
MicroRNAs (miRNAs) reversely modulate gene expression by interacting with the 3′ untranslated region (3′UTR) of messenger RNAs (mRNAs), causing translational repression or degradation of mRNAs [
11,
12]. Dysregulation of miRNAs was associated with AS progression [
13]. We concentrated on the biological significance of miR-328-3p in AS progression, which was predicted to be a candidate downstream miRNA of circ_0004104 by bioinformatic database. Guo et al. claimed that miR-328-3p attenuated ox-LDL-mediated dysfunction in HUVECs [
14]. Nevertheless, the working mechanism of miR-328-3p in AS still needs to be further clarified.
Tripartite motif 14 (TRIM14) is one of the members of TRIM family [
15]. TRIM14 exerted an oncogenic role in many human malignancies [
16‐
19]. In addition, Huang et al. demonstrated that TRIM14 could accelerate the activation of endothelium through activating NF-κB signaling [
20]. TRIM14 was predicted by bioinformatic database to be a downstream gene of miR-328-3p, and the working mechanism of TRIM14 in AS progression was investigated.
We initially explored the role of circ_0004104 in AS cell model. Subsequently, the working mechanism of circ_0004104 was explored through bioinformatic analysis and rescue experiments.
Methods
Cell line
Human umbilical vein endothelial cells (HUVECs) acquired from Chinese Academy of Medical Sciences, Shanghai institute Cell Bank (Shanghai, China) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) plus 10% fetal bovine serum (FBS, Hyclone, Carlsbad, CA, USA) and 1% antibiotics (Gibco) under 37℃ humidified atmosphere with 5% CO2.
AS cell model
HUVECs were exposed to 100 μg/mL ox-LDL (Solarbio, Beijing, China) for 24 h to establish AS cell model as previously reported [
8,
14].
Real-time quantitative polymerase chain reaction (RT-qPCR)
RNA samples were isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using the miScript Reverse Transcription kit (for miRNA; Qiagen, Valencia, CA, USA) and RevertAid First Strand cDNA Synthesis Kit (for circRNA and mRNA; Invitrogen). cDNA was amplified via the SYBR™ Green PCR Master Mix (Invitrogen). The primers purchased from Sangon Biotech (Shanghai, China) were shown in Table
1. Relative abundance of circ_0004104, SPARC and TRIM14 was analyzed using the 2
−ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference, while the fold change of miR-328-3p was calculated using the 2
−ΔΔCt method with U6 as reference.
Table 1
Specific primers in RT-qPCR assay
circ_0004104 | Forward | 5′-AGACCTGTGACCTGGACAATG-3′ |
| Reverse | 5′-GTGCACTTTGTGGCAAAGAA-3′ |
SPARC | Forward | 5′-GGTATCTGTGGGAGCTAATC-3′ |
| Reverse | 5′-CTGGTGGGGTCCTGGCACAC-3′ |
miR-328-3p | Forward | 5′-CCTCTCTGCCCTTCCG-3′ |
| Reverse | 5′-GAACATGTCTGCGTATCTC-3′ |
TRIM14 | Forward | 5′-GAGGTCGGAGCTTGTCGAG-3′ |
| Reverse | 5′-TTCTTGGCTGAGTTTCTGCAC-3′ |
U6 | Forward | 5′-CTCGCTTCGGCAGCACA-3′ |
| Reverse | 5′-AACGCTTCACGAATTTGCGT-3′ |
GAPDH | Forward | 5′-AAGAAGGTGGTGAAGCAGGC-3′ |
| Reverse | 5′-GTCAAAGGTGGAGGAGTGGG-3′ |
Cyclization validation
RNA samples (2 μg) were incubated with 3 U/μg RNase R (Epicentre Technologies, Madison, WI, USA), and RNA levels were determined by RT-qPCR.
Actinomycin D treatment
Transcription inhibitor Actinomycin D (2 mg/mL; Sigma, St. Louis, MO, USA) was added to the culture medium, and RNA levels were examined by RT-qPCR at specific time points.
Oligonucleotides or plasmids transfection
Ectopic expression plasmid of circ_0004104 (circ_0004104), pLCDH-cir empty vector (vector), small interfering RNA against circ_0004104 (si-circ_0004104), negative control of siRNA (si-NC), TRIM14 overexpression plasmid (TRIM14) and empty vector (pcDNA) were purchased from Sangon Biotech, and mimics of miR-328-3p (miR-328-3p), miR-NC, inhibitor of miR-328-3p (anti-miR-328-3p) and anti-miR-NC were acquired from Genepharma (Shanghai, China). All oligonucleotides or plasmids were transfected into HUVECs with Lipofectamine 3000 reagent (Invitrogen).
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay
At specific time points, HUVECs were incubated with MTT reagent (Sigma) for 4 h. Afterwards, a total of 200 μL dimethyl sulfoxide (DMSO; Sigma) was added to dissolve the formazan products after discarding cell supernatant. The absorbance (490 nm) was determined by the microplate reader (Bio-Rad, Hercules, CA, USA).
Flow cytometry
HUVECs were simultaneously stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) of the Cell Apoptosis Detection Kit (Qiagen). The apoptotic percentage of HUVECs was evaluated by the flow cytometer (BD Biosciences, San Jose, CA, USA).
Angiogenic capacity analysis via capillary-like network formation assay
HUVECs were plated onto Matrigel (BD Biosciences)-pre-coated 96-well cell culture plates (3 × 104 cells/well). After culturing for 48 h, the average number of branches of each node was analyzed.
Western blot assay
HUVECs were disrupted using whole cell lysis buffer (Beyotime, Shanghai, China). Protein samples (35 μg) were loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After sealing with 5% bovine serum albumin (BSA; Sangon Biotech), immunoblot assay was applied through incubating the membrane with the diluted primary antibodies and the horse radish peroxidase (HRP) conjugated secondary antibody (Abcam). Immuno-reactive signals were determined by the enhanced chemiluminescent (ECL) chromogenic substrate (Beyotime). The primary antibodies contained anti-Cleaved-caspase 3 (anti-Cleaved-casp3, ab32042, Abcam, Cambridge, MA, USA), anti-vascular endothelial growth factor A (anti-VEGFA, ab52917, Abcam), anti-TRIM14 (SAB1410027, Sigma) and anti-GAPDH (ab8245, Abcam).
Enzyme-linked immunosorbent assay (ELISA)
The culture supernatant of HUVECs was collected to assess the release of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) using commercial Human TNF-α/IL-1β Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).
Determination of cell oxidative stress
Cell oxidative stress was analyzed through measuring the production of superoxide dismutase (SOD) and malondialdehyde (MDA) using their corresponding commercial kits (Jiancheng Biotech, Nanjing, China).
Dual-luciferase reporter assay
The fragment of circ_0004104 or the 3′UTR fragment of TRIM14, including the miR-328-3p-binding sequence, was inserted into psiCHECK2 luciferase plasmid (Promega, Madison, WI, USA) to generate circ_0004104 wt and TRIM14 3′UTR wt. Meanwhile, mutated counterparts were constructed to generate circ_0004104 mut and TRIM14 3′UTR mut. HUVECs were seeded onto 12-well plates and co-transfected with luciferase plasmids and miR-NC or miR-328-3p. After 48-h transfection, the relative luciferase intensities were determined using the Dual-Luciferase Reporter Assay Kit (Promega).
RNA immunoprecipitation (RIP) assay
RIP experiment was employed to confirm the binding relation between circ_0004104 and miR-328-3p with Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cell extracts were prepared using RIP lysis buffer, and anti-Argonaute2 (anti-Ago2; Millipore) or anti-Immunoglobulin G (anti-IgG; Millipore)-pre-coated magnetic beads were incubated with cell lysates. The levels of enriched RNAs were measured by RT-qPCR.
RNA-pull down assay
Cell lysates (2 μg) were incubated with 100 pmol Bio-miR-NC, Bio-miR-328-3p-mut or Bio-miR-328-3p-wt. The reaction mixture was then incubated with 100 μL agarose beads (Millipore) for 1 h. The retrieved RNAs were measured by RT-qPCR.
Statistical analysis
All experiments were repeated for three times. Statistical analysis was carried out using GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA). Data were represented as mean ± standard deviation (SD). The differences were analyzed by Student’s t-test (two groups) or one-way analysis of variance (ANOVA) (more than two groups). Differences were identified as statistically significant with the P value of less than 0.05.
Discussion
AS results in severe cardio/cerebral-vascular disorders, containing coronary heart disease and stroke [
27]. The phenotypic transformation of endothelial cells is an important induction factor in AS initiation and progression [
28]. Hence, it is essential to uncover the crucial molecules involved in the phenotypic transformation of endothelial cells. Dysregulated circRNAs have been associated with AS progression by previous studies [
21,
29]. For instance, circ_0044073 was highly expressed in AS, and it promoted the proliferation and invasion of vascular smooth muscle cells and vascular endothelial cells through sponging miR-107 in AS [
30]. Zhang et al. demonstrated that circ-PTPRA contributed to AS development through up-regulating SP1 via sponging miR-636 [
31]. As for circ_0004104, Wang et al. found that circ_0004104 was highly expressed in coronary artery disease patients compared with controls, and circ_0004104 was identified as a novel bio-marker for the diagnosis of coronary artery disease [
10]. However, the expression pattern and role of circ_0004104 in the pathogenesis of AS remain to be disclosed. Here, we established AS cell model through exposing HUVECs to ox-LDL. Circ_0004104 abundance was markedly up-regulated in ox-LDL-exposed HUVECs relative to un-treated HUVECs. Through using exonuclease RNase R and transcriptional inhibitor Actinomycin D, we found that circ_0004104 was a stable circular transcript. ox-LDL exposure restrained cell viability and tube formation ability and induced the apoptosis, inflammation and oxidative stress status of HUVECs. To analyze the role of circ_0004104 in AS, we overexpressed or silenced circ_0004104 in AS cell model. Circ_0004104 overexpression aggravated ox-LDL-induced injury in HUVECs, whereas ox-LDL-induced damage in HUVECs was largely alleviated by the silencing of circ_0004104, which demonstrated that ox-LDL induced the injury of HUVECs partly through up-regulating circ_0004104.
“MiRNA sponge” mechanism is an important way by which circRNAs function in human diseases [
26,
32]. For example, circ_0010283 accelerated cell viability and motility of ox-LDL-treated vascular smooth muscle cells by sponging miR-370-3p and up-regulating HMGB1 [
24]. Circ-PRMT5 contributed to gastric cancer development through up-regulating MYC via sponging miR-145 and miR-1304 [
33]. To investigate the molecular mechanism by which circ_0004104 functioned in ox-LDL-induced HUVECs, we analyzed circ_0004104-miRNA interactions using bioinformatic database StarBase. MiR-328-3p was predicted as a possible target of circ_0004104 via StarBase database, and their target relation was then confirmed by dual-luciferase reporter assay, RIP assay and RNA-pull down assay. MiR-328-3p was identified as a tumor suppressor in several malignancies through modulating its downstream genes and signal pathways [
34‐
36]. Furthermore, Xing et al. demonstrated that miR-328-3p restrained the proliferation and cell cycle progression of pulmonary artery smooth muscle cells [
37]. Qin et al. demonstrated that miR-328-3p alleviated ox-LDL-induced injury in vascular endothelial cells through reducing FOXO4 abundance in AS [
14]. We found that ox-LDL exposure reduced the level of miR-328-3p in HUVECs. In addition, we found that miR-328-3p was negatively regulated by circ_0004104 in HUVECs. To explore if circ_0004104 functioned through sponging miR-328-3p, we transfected HUVECs with si-circ_0004104 alone or together with anti-miR-328-3p prior to ox-LDL exposure to perform rescue experiments. The results uncovered that circ_0004104 silencing attenuated ox-LDL-mediated injury in HUVECs partly through up-regulating miR-328-3p.
MiRNAs could regulate gene expression through repressing translation or degrading target mRNAs [
12]. StarBase database was utilized to explore the downstream molecules of miR-328-3p. TRIM14 was predicted to be a candidate target of miR-328-3p, and their intermolecular interaction was subsequently verified by dual-luciferase reporter assay. TRIM14 was identified as an oncogene in a variety of cancers. For instance, TRIM14 accelerated the migration and invasion abilities of colorectal cancer cells through regulating SPHK1/STAT3 signaling [
38]. TRIM14 elevated the drug resistance of glioma cells through regulating Wnt/β-catenin signaling [
39]. Huang et al. found that TRIM14 accelerated the activation of endothelium via activating NF-κB pathway [
21]. We found that ox-LDL exposure up-regulated the expression of TRIM14 at mRNA and protein levels. We also found that TRIM14 was negatively regulated by miR-328-3p in HUVECs. We found that circ_0004104 positively regulated TRIM14 expression through acting as miR-328-3p sponge. To explore if miR-328-3p regulated the biological phenotypes of HUVECs via targeting TRIM14, we performed compensation experiments. The results revealed that miR-328-3p protected HUVECs against ox-LDL-mediated damage in HUVECs partly through down-regulating TRIM14.
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