Protective effect of microRNA‑381 against inflammatory damage of endothelial cells during coronary heart disease by targeting CXCR4.

Coronary heart disease (CHD) is the leading cause of human morbidity and mortality worldwide. MicroRNA (miRNA) profiling is an innovative method of identifying biomarkers for many diseases and may be a powerful tool in the diagnosis and treatment of CHD. The present study aimed to analyze the effects of miRNA (miR)‑381 on the inflammatory damage of endothelial cells during CHD. A total of 21 patients with CHD and 21 healthy control patients were enrolled in this study. Reverse transcription‑quantitative PCR, western blotting and immunofluorescence assays were conducted to examine the expression levels of miR‑381, C‑X‑C chemokine receptor type 4 (CXCR4), Bcl‑2, Bax, Cleaved‑Caspases‑3 and ‑9, p38, ERK1/2 and JNK. Cell Counting Kit‑8, EdU and flow cytometry experiments were performed to evaluate cell proliferation and apoptosis. An ELISA was adopted to determine the expressions of inflammatory factors (interleukins‑8, ‑6 and ‑1β, and tumor necrosis factor‑α). In addition, a dual‑luciferase reporter assay was used to determine the relationship between miR‑381 and CXCR4. Decreased miR‑381 expression and increased CXCR4 expression in the plasma were observed in the CHD group compared with the normal group, which indicated a negative relationship between miR‑381 and CXCR4. Overexpression of miR‑381 significantly promoted the proliferation and inhibited the apoptosis of oxidized low‑density lipoprotein (OX‑LDL)‑induced human umbilical vein endothelial cells (HUVECs) through mitogen‑activated protein kinase pathway by targeting and inhibiting CXCR4. Furthermore, overexpression of miR‑381 reduced the release of inflammatory factors in OX‑LDL‑induced HUVECs. By contrast, reduced expression of miR‑381 exerted the opposite effects, which were subsequently reversed by silencing CXCR4 expression. Results from the present study indicated that miR‑381 was a CHD‑related factor that may serve as a potential molecular target for CHD treatment.

Introduction

Coronary heart disease (CHD) is a common cardiovascular disease that endangers human health and has become the primary cause of death in adult worldwide. It has been reported ~350,000 patients with CHD succumb to mortality every year (1,2). CHD is one of the most common types of atherosclerotic organ disease (3). Many atherosclerotic processes are closely related to the occurrence of CHD, such as inflammation, oxidative stress, hemodynamic changes and endothelial cell damage (4). Structural and functional damage to endothelial cells leads to the development of atherosclerosis, long-term hyperlipidemia, hemodynamic changes and inflammatory response, which result in endothelial dysfunction, lipid invasion of the arterial media, and gradually evolving into fibrous atherosclerotic plaques, eventually leading to CHD (5,6). Currently, the main treatment methods of CHD include drug therapy, percutaneous coronary intervention and coronary artery bypass grafting (7).

MicroRNAs (miRNAs) are a class of non-coding small RNAs, 18–22 nucleotides in length, that can bind to the 3′-untranslated region (UTR) of a target mRNA at the post-transcriptional level by base-pairing to inhibit translation and to regulate the expression of the corresponding target genes (8,9). A recent study has reported that miRNAs can bind to 5′-terminal region of the target mRNA and activate the expressions of target genes (10). miRNAs are involved in cell differentiation, growth, development and apoptosis (11,12) and have been found to be widely involved in the regulation of various vascular diseases, including vascular inflammation, coronary artery disease, myocardial infarction and heart failure, and atherosclerosis (13). For example, miRNA (miR)-142-3p was demonstrated to suppress myocardial cell injury induced by hypoxia/reoxygenation (14). miR-200c affects the mitogen-activated protein kinase (MAPK) pathway signaling pathway and promotes cardiomyocyte hypertrophy by targeting dual-specificity phosphatase 1 (15). A recent study reported that miRNAs serve an important regulatory role in the occurrence and development of CHD (16). In a separate study, the levels of miRNAs in plasma samples were compared in 69 patients with CHD and 30 healthy controls (17). The levels of miR-145, miR-155 and miR-let-7c were significantly reduced in plasma of patients with CHD. miR-145, miR-155 and miR-let-7c are considered to be independent risk factors for CHD and can be used as biomarkers for detection of CHD (17). In addition, miR-132, miR-140-3p and miR-210 have been reported to accurately predict cardiovascular mortality in patients with acute coronary syndrome, which can be used as prognostic markers in patients with CHD (18). It has been demonstrated that miR-146 can be used as an independent predictor of coronary collateral circulation; increased expression in plasma is positively correlated with good collateral circulation (19). A tumor-suppressing role of miR-381 was reported in various cancers, including breast cancer, osteosarcoma and ovarian cancer (20–22). In these cancers, the levels of miR-381 are commonly downregulated. It has been shown that downregulation of miR-381 in colon cancer induces proliferation and invasion of colon cancer cells (23). miR-381 expression is inversely related to the expression of multidrug resistant protein 1 gene and serves an important role in multidrug resistance (24). Results from the aforementioned studies indicate that miR-381 is closely related to the occurrence and development of human disease. Data from the present study indicated that the expression of miR-381 is decreased in the plasma of patients with CHD. Considering the abnormal expression of miR-381, it was hypothesized that dysregulation of miR-381 may serve as a biomarker of CHD. Consistently, it was observed that overexpression of miR-381 promotes the proliferation of oxidized low-density lipoprotein (OX-LDL)-induced human umbilical vein endothelial cells (HUVECs) through the MAPK pathway by targeting and inhibiting chemokine receptor 4 (CXCR4) expression, which demonstrated that miR-381 may be a CHD-related factor.

CXCR-4, a 7-transmembrane G protein-coupled chemokine receptor, is widely expressed by various cells, including cells in the immune and central nervous system, progenitor cells in the bone marrow and mononuclear cells (25). Particularly, CXCR4 has been reported to be expressed mainly at the plasmalemma of cardiac myocytes (26). It has been demonstrated that vascular CXCR4 could prevent atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function (27). CXCR4 serves an important role in coronary artery development (28); a high level of CXCR4 in peripheral CD34+ cells is linked to good coronary collateralization in patients with chronic total coronary occlusion.

OX-LDL is an oxidative product of native LDL and is involved in a wide variety of biological activities, including vascular endothelial injury and coagulation disorders (29). OX-LDL has been related with atherosclerotic plaques and reported to serve a role in atherogenesis (30). OX-LDL-induced endothelial cell dysfunction serves an important role in the pathogenesis of cardiovascular diseases (31). The present study attempted to investigate the role of miR-381 in regulation of CHD risk and its possible mechanism in relation to the MAPK signaling pathway in OX-LDL-induced endothelial cells.

Materials and methods

Plasma samples

Plasma samples (n=21) from the aortas of patients with CHD and normal plasma samples (n=21) from healthy control patients were collected from the Department of Cardiology of Nanjing Chest Hospital (Nanjing, China) from March 2017 to January 2018. The CHD group comprised 9 males and 12 females (age, 46–79 years). The inclusion criteria were based on the CHD diagnostic criteria (32): Patients with chest discomfort and/or ischemic ST-T changes as determined by an electrocardiograph, and coronary angiography showing the four major coronary arteries of the left main, left anterior descending, left circumflex and right coronary artery, with at least one artery containing pathological changes of stenosis ≥50%. The normal control group comprised 10 males and 11 females (age, 41–75 years). All participants provided written informed consent before samples were collected, and the study was approved by the Institutional Medical Ethics Committee of Nanjing Chest Hospital. A total of 5 ml of arterial blood was collected from patients in each group before coronary arteriography. Blood samples were collected in EDTA tubes, and centrifuged (2,500 × g) at 4°C for 10 min to isolate the plasma. Plasma was stored at −80°C before following measurement.

Cell culture and transfection

HUVECs were purchased from American Type Culture Collection and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C with 5% CO2. HUVECs were treated with 100 µg/ml OX-LDL for 24 h for the subsequent experiments.

HUVECs (1×105/well) were transiently transfected with 50 nM of miR-381 mimics, miR-381 inhibitors, negative control (NC) oligonucleotides, small interfering RNA (siRNA)-CXCR4 or the related negative control (siRNA-NC; all Shanghai GenePharma Co., Ltd.) using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 30 min, according to the manufacturer's instructions. The sequences were as below: miR-381 mimics, forward 5′-CCAGAUCGUAAGUGGUACCGUU-3′ and reverse 5′-CUCUACACCGAACUAUAUCAGU-3′; miR-381 inhibitors, 5′-TATCCGACTTGTAGCATTAACT-3′; NC, forward 5′-GAGGACAUUUCUGUCGAACAA-3′ and reverse 5′-AAGCACUAUUCCAAUGUGCUG-3′; siRNA-NC: 5′-GGTGGTCTATGTTGGCGTCTG-3′; si-CXCR4 sense: 5′-GATCCCGGGTGGTCTATGTTGGCGTCTGGAAGCTTGCAGACGCCAACATAGACCACCTTTTTT-3′, antisense: 5′-CTAGAAAAAAGGTGGTCTATGTTGGCGTCTGCAAGCTTCCAGACGCCAACATAGACCACCCGG-3′. All RNA vectors were labeled with green fluorescence protein (GFP). Transfection efficiency was determined by GFP expression and reverse transcription-quantitative PCR (RT-qPCR) 24 h later. After 48 h, transfected cells were collected for further experiments.

RT-qPCR

HUVECs were seeded (2×105 cells/well) into 6-well plates 12 h prior to transfection. OX-LDL (Hangzhou Union Biotechnology Co., Ltd.) was added to the cells 24 h following transfection, then the cells were incubated at 37°C for another 48 h. After washing twice with PBS, total RNA was isolated from cells or 1 ml plasma using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized from total RNA using a RevertAid™ First-strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. RT-qPCR was carried out with the reagents of an SYBR Premix Ex Taq kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was run in triplicate at 95°C for 2 min followed by 40 cycles of 95°C for 5 sec, 55°C for 45 sec and 75°C for 25 sec. Comparative quantification was performed using the 2−ΔΔCq method (33). U6 or GAPDH was used as an endogenous control for miR-381 or CXCR4, respectively. The sequences of primers were as follows: GAPDH, forward 5′-CGGAGTCAACGGATTTGGTCGTAT-3′, reverse 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′.

Dual-luciferase assay

Bioinformatical analysis was performed using TargetScan 7.1 (http://www.targetscan.org/vert_71). Wild-type and mutant CXCR4 3′-UTR dual-luciferase reporter vectors were constructed by sub-cloning the human CXCR4 mRNA 3′-UTR and mutant 3′-UTR sequences into pmirGLO Dual-Luciferase Reporter vectors (Promega Corporation). Mutations in the miR-381 target site of the 3′-UTR of CXCR4 were generated using a QuickChange Site-Directed Mutagenesis kit (GenScript). 293 cells (American Type Culture Collection) were transfected with 80 ng luciferase reporter vectors and miR-381 mimics or mimics NC using the Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h, luciferase activities were measured using Dual-Luciferase Reporter System (Berthold Detection Systems GmbH) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.

Western blotting

HUVECs were seeded (2×105 cells/well) into 6-well plates 12 h prior to transfection. OX-LDL (100 µg/ml) was added to the cells 24 h following transfection, then the cells were incubated for another 48 h at 37°C. After washing twice with PBS, the cells were lysed in RIPA lysis buffer (Cell Signaling Technology, Inc.). The Protein concentration was detected using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Proteins (20 µg/lane) were extracted from cells and separated on a 12% SDS-PAGE gel. Proteins were transferred to PVDF membranes (EMD Millipore), which were subsequently blocked with 5% skimmed milk for 2 h at room temperature and incubated with the primary antibodies overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. All antibodies used were purchased from Cell Signaling Technology, Inc. and details are shown in Table I. Protein expression levels were normalized to the internal control GAPDH and visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc.). The relative intensities of protein blots were quantified using ImageJ software (version 1.48; National Institutes of Health).

Table I.

Antibodies used in this study.

Name	Cat. no.	Dilution
CXCR4	97680	1:500
Cleaved-Caspase-3	9661	1:500
Cleaved-Caspase-9	9505	1:500
Bcl-2	2872	1:500
Bax	2774	1:500
p38	8690	1:500
p-p38	4511	1:500
ERK	4695	1:500
p-ERK	4370	1:500
JNK	9252	1:500
p-JNK	9255	1:500
GAPDH	8884	1:2,000
Anti-mouse IgG (HRP)	7076	1:2,000
Anti-Rabbit IgG (HRP)	7074	1:2,000
H&L Alexa Fluor® 488	ab150077	1:300

All antibodies were from Cell Signaling Technology, Inc., with the exception of ab150077 (Abcam). Bax, Bcl-2-like protein 4; Bcl-2, B-cell lymphoma 2; ERK, extracellular signal-regulated kinase; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; p-, phosphorylated.

Cell Counting Kit-8

Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. Briefly, HUVECs were seeded (1×104 cells/well) into 96-well plates 12 h prior to transfection. OX-LDL (80 µg/ml) was added to the cells 24 h following transfection and incubated at 37°C for 24, 48 and 72 h, respectively. At each of the three time points, 10 µl of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

EdU assay

The EdU assay was performed to evaluate the effects of miR-381 on cell proliferation. HUVECs were seeded (1×104 cells/well) into 96-well plates 12 h prior to transfection. OX-LDL (80 µg/ml) was added to the cells 24 h following transfection and incubated for another 48 h at 37°C. EdU (100 µl) was added and cells were incubated at 37°C for another 8 h. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized with Triton X-100, and blocked with PBS containing 10% goat serum for 1 h at 25°C. Cells were stained by Cell-Light™ EdU Apollo® 488 In Vitro Imaging kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The images were captured under an IX53 fluorescent microscope (magnification, ×200; Olympus Corporation). EdU-positive cells were counted in three randomly selected fields.

Immunofluorescence staining

HUVECs were cultured on 14-mm-diameter poly-L-lysine-coated cover slides, then treated with OX-LDL as described above. Then, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and treated with 0.2% Triton X-100 at room temperature for 10 min. Afterwards, cells were incubated with anti-CXCR4 antibody at 4°C overnight, followed by incubation with an Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (1:300; cat. no. ab150077; Abcam) for 1 h at room temperature. Nuclei were stained with DAPI for 10 min at room temperature. Cells were visualized within three randomly selected fields under a laser scanning confocal microscope (SP8; Leica Microsystems GmbH).

ELISA

HUVECs were seeded (1×104 cells/well) into 96-well plates 12 h prior to transfection. OX-LDL (80 µg/ml) was added to the cells 24 h following transfection and incubated for another 48 h. The cell supernatant was collected, interleukin (IL)-8 (cat. no. KHC0081), IL-6 (cat. no. BMS213HS), IL-1β (cat. no. BMS224-2) and tumor necrosis factor (TNF)-α (cat. no. BMS223HS) were measured using commercial ELISA kits (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocols.

Cell apoptosis

Apoptosis was evaluated using an Annexin V-FITC/Propidium iodide (PI) staining kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacture's protocol. HUVECs were seeded in 6-well plates at the density of 2×105 cells/well. After cell transfection followed by OX-LDL treatment for 24 h as aforementioned, cells were washed in PBS and resuspended in binding buffer. Then, cells were labeled with Annexin V-FITC and PI in the dark for 20 min at room temperature. Apoptosis was detected by FlowJo v10 (FlowJo LLC) using a FACSCalibur flow cytometer (BD Biosciences).

Statistical analysis

All the results are presented as mean ± SEM, and each test was repeated at least three times. All statistical analyses were carried out with the GraphPad Prism 5.0 software (GraphPad Software, Inc.) using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Relative expressions of miR-381 and CXCR4 in the plasma of the normal and CHD groups

RT-qPCR was conducted to evaluate the expression levels of miR-381 and CXCR4 in the plasma of the normal and CHD groups. The result demonstrated that, the expression level of miR-381 in the plasma of patients with CHD was significantly decreased, whereas that of CXCR4 was notably increased compared with the normal group (Fig. 1A and B).

Figure 1.

Relative expression levels of miR-381 and CXCR4 in the plasma of patients in the normal and CHD groups. (A) RT-qPCR assay was performed to evaluate the expression of miR-381 in in the plasma of the normal and CHD groups. (B) RT-qPCR assay was performed to evaluate the expression of CXCR4 in the plasma of the normal and CHD groups. *P<0.05 vs. Normal. CHD, coronary heart disease; CXCR4, C-X-C chemokine receptor type 4; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

CXCR4 is a target of miR-381

HUVECs were used in the present study to mimic the inflammatory damage of endothelial cells during CHD. To investigate the molecular function of miR-381 in HUVECs, cells were successfully transfected with miR-381 mimics or miR-381 inhibitors (Fig. 2A). The successful transfection of miR-381 mimics or inhibitors was also confirmed by fluorescence microscopy (Fig. 2B). Similarly, siRNA-CXCR4 remarkably downregulated the expression of CXCR4 compared with the siRNA-NC-transfected group in HUVECs (Fig. 2C).

Figure 2.

CXCR4 is a target gene of miR-381. (A) RT-qPCR assay was performed to evaluate the expression of miR-381 to confirm the successful transfection of miR-381 mimics or miR-381 inhibitors. (B) Laser scanning confocal microscopy was employed to detect the transfection by observing green fluorescence protein (magnification, ×200). (C) RT-qPCR was performed to evaluate the levels of CXCR4 in cells transfected with siRNA-CXCR4. (D) TargetScan was used to predict the target genes of miR-381; a putative miR-381 binding site was identified in the 3′UTR of CXCR4 (highlighted in red in the MUT sequence). (E) To determine if CXCR4 was a direct target of miR-381, a dual-luciferase reporter assay was performed. (F) Western blotting and (G) RT-qPCR were used to test the protein and mRNA expression levels, respectively, of CXCR4 in cells transfected with miR-381 mimics or miR-381 inhibitors. (H) RT-qPCR assay determination of the expression of miR-381 in cells transfected with siRNA-CXCR4. *P<0.05 vs. the corresponding NC group. CXCR4, C-X-C chemokine receptor type 4; miR, microRNA; MUT, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; UTR, untranslated region; WT, wild-type.

TargetScan was used to predict the target genes of miR-381. The result demonstrated that the 3′UTR of CXCR4 contained a potential miR-381 target site (Fig. 2D). Further, the result of luciferase reporter gene activity assay demonstrated that miR-381 mimics markedly decreased the relative luciferase activity in cells transfected with wild-type CXCR4 3′UTR-WT, whereas the luciferase activity had no significant change in cells co-transfected with miR-381 mimics and mutant CXCR4 3′UTR-MUT (Fig. 2E).

The results of western blotting and RT-qPCR assay suggested that overexpression of miR-381 inhibited the protein and mRNA expressions of CXCR4, respectively, whereas inhibition of miR-381 notably increased the protein and mRNA expressions of CXCR4 in HUVECs (Fig. 2F and G). In addition, downregulation of CXCR4 significantly increased the expression of miR-381 in HUVECs (Fig. 2H).

Effects of miR-381 on OX-LDL-induced cell proliferation

In HUVECs, RT-qPCR results demonstrated that OX-LDL treatment significantly decreased the expression of miR-381 and increased the mRNA expression level of CXCR4 (Fig. 3A and B). In addition, western blotting and immunofluorescence assays indicated that the expression of CXCR4 significantly increased in OX-LDL-induced HUVECs compared with untreated controls (Fig. 3C and D).

Figure 3.

Expressions of miR-381 and CXCR4 in OX-LDL-induced HUVECs. RT-qPCR assay tested the expression levels of (A) miR-381 and (B) CXCR4. (C) Western blotting and (D) immunofluorescence assays were performed to analyze the protein expression level of CXCR4 in OX-LDL-induced HUVECs (magnification, ×200). *P<0.05 vs. Blank. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; OX-LDL, oxidized low-density lipoprotein; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

A CCK-8 assay was performed to detect the effect of miR-381 and CXCR4 on the proliferation of OX-LDL-induced cells. The result suggested that miR-381 mimics notably increased OX-LDL-induced cell proliferation of HUVECs (P<0.05), while inhibition of miR-381 significantly decreased OX-LDL-induced cell proliferation of HUVECs for 24, 48 and 72 h (P<0.05), respectively (Fig. 4A). Downregulation of CXCR4 by siRNA significantly promoted OX-LDL-induced cells proliferation (P<0.05), whereas co-treatment with siRNA CXCR4 notably ameliorated the effect of miR-381 inhibitors (Fig. 4B). An EdU assay was performed, and the results indicated that overexpression of miR-381 and inhibition of CXCR4 could both significantly promote the OX-LDL-induced proliferation of HUVECs (Fig. 4C). In addition, siRNA CXCR4 could improve the effect of miR-381 inhibition on OX-LDL-induced HUVEC cell proliferation (Fig. 4C).

Figure 4.

Effects of miR-381 on OX-LDL-induced cell proliferation. (A and B) Cell Counting Kit-8 assay was performed to detect the proliferation of OX-LDL-induced HUVECs. *P<0.05 vs. Blank or miR-381 inhibitiors. (C) EdU assay was performed to detect the proliferation of OX-LDL-induced HUVECs (magnification, ×200). *P<0.05. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA.

Effects of miR-381 on inflammatory cytokine release from OX-LDL-induced HUVECs

ELISA was used to detect the levels of IL-8, IL-6, IL-1β and TNF-α in the cell supernatant. The results demonstrated that miR-381 mimics significantly reduced the levels of IL-8, IL-6, IL-1β and TNF-α in OX-LDL-induced HUVEC cell supernatant, whereas inhibition of miR-381 expression significantly promoted the expression levels of IL-8, IL-6, IL-1β and TNF-α in OX-LDL-induced cell supernatant of HUVECs. Furthermore, siRNA CXCR4 transfection significantly suppressed the levels of IL-8, IL-6, IL-1β and TNF-α in OX-LDL-induced cell supernatant of HUVECs, and could suppress the promotive function of miR-381 inhibitors (Fig. 5).

Figure 5.

Effects of miR-381 on inflammatory cytokine release in OX-LDL-induced cells. ELISA was used to detect the levels of IL-8, IL-6, IL-1β and TNF-α in the cell supernatant. *P<0.05. CXCR4, C-X-C chemokine receptor type 4; IL, interleukin; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA; TNF, tumor necrosis factor.

Effects of miR-381 on the cell apoptosis of OX-LDL-induced HUVECs

Flow cytometry was used to evaluate apoptosis of OX-LDL-induced HUVECs. The scatterplots in Fig. 6A are divided into four quadrants: Q1, Q2, Q3 and Q4. Q1 (FITC−/PI+) represents necrotic cells, Q2 (FITC+/PI+) represents late apoptotic cells, Q3 (FITC+/PI−) represents early apoptotic cells and Q4 (FITC−/PI−) represents living cells. The percentage of early apoptotic cells was lower in miR-381 mimics-treated cells (5.49%) compared with miR-381 mimics NC treated cells (13.0%; P<0.05). Conversely, transfection of miR-381 inhibitors increased the proportion of early apoptotic cells from 13.3% of cells transfected with inhibitors NC to 27.9% (P<0.05). Only a small percentage of cells (0–5%) in both miR-381 mimics and inhibitors-transfected cells were FITC−/PI+, which suggested that necrotic cell death was not an acting mechanism in the observed phenomenon. In addition, downregulation of CXCR4 decreased the apoptosis of OX-LDL-induced HUVECs; conversely, inhibition of miR-381 abolished these inhibitory effects. Together, these findings confirmed that miR-381 suppressed cell apoptosis of OX-LDL-induced HUVECs, and inhibition of miR-381 promoted early apoptosis.

Figure 6.

Effects of miR-381 on cell apoptosis of OX-LDL-induced HUVECs. (A) Flow cytometry was used to evaluate the apoptosis of OX-LDL-induced HUVECs transfected with miR-381 mimics, mimics NC, inhibitors, inhibitors NC, siRNA CXCR4, siRNA NC or miR-381 inhibitors + siRNA CXCR4. *P<0.05 vs. NC. (B) Western blotting assay was performed to evaluate the protein expression levels of apoptotic-related proteins, including Bcl-2, Bax, Cleaved-Caspase-3 and Cleaved-Caspase-9. *P<0.05 vs. blank. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA.

Western blotting assay was performed to evaluate the expression levels of apoptotic-related proteins, including B-cell lymphoma 2 (Bcl-2), Bcl-2-like protein 4 (Bax), Cleaved-Caspase-3 and Cleaved-Caspase-9 (Fig. 6B). The results indicated that miR-381 mimics and siRNA CXCR4 transfections significantly inhibited the expression levels of pro-apoptotic proteins Bax, Cleaved-Caspase-3 and Cleaved-Caspase-9, and promoted the expression of anti-apoptotic protein Bcl-2. Downregulation of miR-381 promoted the expression of pro-apoptotic proteins (Bax, Cleaved-Caspase-3 and Cleaved-Caspase-9) and reduced the expression of the anti-apoptotic protein Bcl-2, suggesting that downregulation of miR-381 promoted cell apoptosis; however, co-transfection with siRNA CXCR4 partially attenuated these effects (Fig. 6B).

Effects of miR-381 on MAPK signaling pathway

Western blotting was conducted to determine the protein expression levels of MAPK signaling pathway-related proteins in OX-LDL-induced HUVECs, including phosphorylated (p-)p38, p38, extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK) and p-JNK (Fig. 7). The results demonstrated that, compared with the blank group, the protein expressions of p-p38, p-ERK and p-JNK were notably decreased in the miR-381 mimics group and in the siRNA CXCR4 group in OX-LDL-induced HUVECs, whereas inhibition of miR-381 promoted the expression levels of p-p38, p-ERK and p-JNK, and the levels of total proteins, including p38, ERK and JNK, had no obvious change. In addition, co-transfection with miR-381 inhibitors and siRNA CXCR4 could ameliorate the promoting effect of miR-381 inhibitors in OX-LDL-induced HUVECs (Fig. 7).

Figure 7.

Effects of miR-381 on MAPK signaling pathway. Western blotting was conducted to determine the protein expression levels of MAPK signaling pathway-related proteins (p-p38, p38, p-ERK, ERK, p-JNK and JNK) in OX-LDL-induced HUVECs transfected with miR-381 mimics, mimics NC, inhibitors, inhibitors NC, siRNA CXCR4, siRNA NC or miR-381 inhibitors + siRNA CXCR4. *P<0.05 vs. Blank. CXCR4, C-X-C chemokine receptor type 4; ERK, extracellular signal-regulated kinase; HUVECs, human umbilical vein endothelial cells; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; p-, phosphorylated; siRNA, small interfering RNA.

Discussion

CHD is a major cause of mortality in the world (34). Endothelial cells are involved in a number of inflammatory diseases, including psoriasis, diabetes, cancer and rheumatoid arthritis (35). A previous study identified that chronic inflammation is associated with increased atherosclerosis, accelerated cardiovascular mortality and morbidity (36). Endothelial dysfunction is considered to be one of the important causes of cardiovascular diseases (37). The present study examined the potential role of miR-381 and CXCR4 in endothelial cells in CHD.

A previous study demonstrated that miRNAs serve important roles in many biological activities, including cell proliferation, inflammation and apoptosis (38). In CHD, miR-423, miR-23 and miR-199a have been reported to serve as biomarkers for therapeutic targets (39–41). miR-381 has been reported to be dysregulated in various cancers (20–22), but few studies have focused on the role of miR-381 in CHD. A recent study demonstrated that overexpression of miR-381 in RAW264.7 cell lines decreased the concentrations of IL-1β and TNF-α (42). In addition, miR-381 can reduce inflammation and the infiltration of macrophages by targeting high-mobility group box 1 mRNA (43). In addition, miR-381 can regulate Notch signaling-mediated cardioprotective effect in cardiomyocytes (44). In the present study, the expression of miR-381 in the plasma of patients with CHD was significantly lower compared with the expression levels in healthy control patients, which suggested that miR-381 may serve an important role in the process of CHD.

Endothelial cell structural and functional damage occurs at the beginning of CHD (5). Endothelial cells are the inner layers of vascular walls and are important regulators of vascular inflammation, blood aggregation, vascular tension and capillary permeability (45). Resting endothelial cells express no or low levels of adhesion molecules and are resistant to leukocyte recruitment. However, various CHD stimulants, including TNF-α, IL-1β or OX-LDL, activate the endothelial inflammatory cascade (41). The present study revealed that overexpression of miR-381 significantly promoted OX-LDL-induced HUVEC proliferation, decreased apoptosis and suppressed the inflammation response, whereas downregulation of miR-381 exhibited the opposite effect.

MAPK comprises three main pathways, ERK1/2, JNK and p38 cascades (46). MAPKs serve an important intracellular signaling role in response to extracellular stimuli (46). Activated MAPKs phosphorylate and activate transcription factors in the cytoplasm or nucleus (47). A recent study demonstrated that MAPK signaling pathway affects the development of coronary artery disease (48). Activated MAPK signaling pathway could regulate the expressions of cytokines and microRNAs in coronary artery disease (49). The present study demonstrated that miR-381 may regulate MAPK signaling pathway in OX-LDL-induced HUVECs by targeting CXCR4.

In summary, the present study demonstrated that low miR-381 expression may contribute to high CXCR4 expression and protected the endothelial cells against inflammatory damage through the MAPK signaling pathway during CHD. Therefore, miR-381 could be a potential target for the treatment of CHD.