Long Noncoding RNA EZR-AS1 Regulates the Proliferation, Migration, and Apoptosis of Human Venous Endothelial Cells via SMYD3

Numerous studies have shown that long noncoding RNAs (lncRNAs) play essential roles in the development and progression of human cardiovascular diseases. However, whether lncRNA ezrin antisense RNA 1 (EZR-AS1) is associated with the progression of coronary heart disease (CHD) remains unclear. Accordingly, the aim of the present study was to evaluate the role of lncRNA EZR-AS1 in patients with CHD and in human venous endothelial cells (HUVECs). The findings revealed that lncRNA EZR-AS1 was highly expressed in the peripheral blood of patients with CHD. In vitro experiments showed that the overexpression of EZR-AS1 could enhance proliferation, migration, and apoptosis by upregulating the expression of EZR in HUVECs; downregulation of lncRNA EZR-AS1 resulted in the opposite effect. lncRNA EZR-AS1 was also found to regulate SET and MYND domain-containing protein 3 (SMYD3), a histone H3 lysine 4-specific methyltransferase, which subsequently mediated EZR transcription. Collectively, these results demonstrate that lncRNA EZR-AS1 plays an important role in HUVECs function via SMYD3 signaling.


Introduction
As a result of economic development and an aging population, the risk of developing cardiovascular disease has gradually increased [1,2]. Coronary heart disease (CHD) is a common, high rate of mortality disease with global influence [3,4], and it is therefore necessary to optimize treatment and prevention strategies.
Ezrin (EZR) is a member of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins, which connects the actin cytoskeleton to the plasma membrane. EZR plays a vital role in numerous processes associated with normal cell growth, including adhesion, cell polarity, and migration [12,13]. SET and MYND domain-containing protein 3 (SMYD3) is a member of the SET and MYND domain (SMYD) family. Studies have shown that SMYD3 interacts with the specific region of trimethylation of histone H3 lysine 4-(H3K4Me3-) modified histone tails, which contributes its recruitment to the key promoter regions of downstream genes and activates the gene expression [14,15]. Further studies indicate that SMYD3 could be recruited to SMYD3 binding sites, which present in the downstream of the EZR promoter, causing local accumulation of SMYD3 and accompanied H3K4Me3 at the EZR gene [16]. lncRNA EZR antisense RNA 1 (EZR-AS1) is 362 bp in length and is located on chromosome 6q25. 3. The expression of EZR-AS1 may promote cell migration and mediate cancer cell differentiation [17]. Despite these discoveries, the role of lncRNA EZR-AS1 in noncancerous pathologies, particularly cardiovascular diseases, remains unclear.
Therefore, the aim of the present study was to determine the function of EZR-AS1 in CHD, by assessing the proliferation, migration, and apoptotic rates of human venous endothelial cells (HUVECs) following the knockdown and overexpression of lncRNA EZR-AS1.

Clinical Blood Samples.
In the present study, 35 patients (24 men and 11 women; 50-75 years of age) were recruited, who had been angiographically diagnosed with CHD at the Guizhou Provincial People's Hospital (Guiyang, Guizhou, China) between June 2018 and August 2018. In addition, 38 individuals without CHD (22 men and 16 women; 50-75 years of age) were enrolled as controls. The inclusion criteria included patients with stable angina pectoris exhibiting ≥1 major coronary artery with >80% stenosis. The exclusion criteria included patients with (i) unstable angina or myocardial infarction; (ii) CHD complicated with other organic heart diseases; and (iii) CHD combined with severe liver disease, kidney diseases, familial hypercholesterolemia, malignant tumors, or inflammatory diseases. All blood samples were immediately stored at 4°C, and RNA was extracted on the same day. The study protocol was approved by the Human Ethics Committee Review Board of the Guizhou Provincial People's Hospital, and oral informed consent was obtained from each patient (Ethics approval No. (2019)068).

Cell Counting
Kit-8 (CCK-8) Assay. HUVECs were seeded into 96-well culture plates at a density of 3 × 10 3 cells/well. The following day, the HUVECs were transfected with the corresponding siRNAs or overexpression pcDNA for 24, 48, and 72 h. Cell viability was assessed using the CCK-8 assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol; 10 μl CCK-8 solution was added to each well, and the plates were incubated for 2 h. The absorbance was then measured at 450 nm using a plate reader.
2.4. Wound Healing Assay. HUVECs were plated in 6-well cell culture plates, and 48 h after transfection, the cell monolayers were scratched with a 200 μl plastic pipette tip. After washing twice with phosphate-buffered saline (PBS), the cells were incubated for 24 h in low-serum medium. The wound closure distance was then determined using an inverted microscope (Olympus Corporation) at ×100 magnification, and the area of closure between the 0 and 24 h time points was calculated.  Figure 1: Expression of lncRNA EZR-AS1 and EZR in peripheral blood. Reverse transcription-quantitative PCR was used to detect the expression levels of (a) lncRNA EZR-AS1 and (b) EZR in the peripheral blood of control subjects and patients with CHD. All results were expressed as mean ± SD. * * P < 0:01 vs. controls.  Figure 2: Effects of lncRNA EZR-AS1 on human venous endothelial cell proliferation, migration, and apoptosis. Cells were transfected with (a) lncRNA si-EZR-AS1 (knockdown) or si-control and with pcDNA-EZR-AS1 (overexpression) or pcDNA-control. Untreated cells were used as a control. Posttransfection with si-EZR-AS1 or pcDNA-EZR-AS1, (b) viability, (c, d) migratory ability, and (e) apoptotic rates of the cells were detected by CCK-8 assay, wound healing assay, Transwell assay, and flow cytometry, respectively. Wound healing was quantified, and the number of migratory cells was counted. Magnification, ×100 (migration). All results were expressed as mean ± SD. * P < 0:05 and * * P < 0:01 vs. si-control; # P < 0:05 and ## P < 0:01 vs. pcDNA-control. 3 BioMed Research International 2.5. Transwell Assay. The cells (5 × 10 4 /plate) were resuspended in 0.1 ml serum-free culture medium and seeded into the upper chamber of a Transwell insert (8 μm pore size, 24well; Corning Inc.). In the lower chamber, 0.8 ml complete culture medium was added as a chemoattractant. Following incubation at 37°C for 48 h, the cells that had migrated to the lower surface were fixed with methanol for 20 min and then stained with 0.1% crystal violet solution for a further 20 min. Finally, the stained cells were counted under an inverted microscope (Olympus Corporation) at ×100 magnification.

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2.6. Flow Cytometry. HUVECs were cultured in 6-well plates to 70-90% confluence. The cells were then digested using trypsin and collected by centrifugation (1000 rpm at 4°C for 5 min). Following two washes with precooled PBS, the cells were collected and resuspended in 500 μl binding buffer (1X); 5 μl Annexin V-APC and 5 μl 7-AAD (Nanjing KeyGen Biotech Co., Ltd.) were added, and the cells were incubated at room temperature in the dark for 15 min. Apoptotic cells were then analyzed using a FACScan flow cytometer (BD Biosciences).

Reverse Transcription-Quantitative PCR (RT-qPCR).
The extraction of RNA from the peripheral blood samples was conducted using Total RNA Extraction Kit of blood according to the manufacturer's instructions (BioTeke Corporation). Total RNA was isolated from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was then reverse transcribed into first-strand cDNA using the PrimeScript RT Reagent Kit (Takara Bio, Inc.), and the expression levels of the target RNA were determined        no. AB-P-R001; 1 : 1,000; Goodhere, Inc.).

Statistical Analysis. Each experiment was repeated ≥3
times, and numerical data are presented as mean ± SD. The differences between the means were analyzed using Student's unpaired t-tests or one-way ANOVA where appropriate. All statistical analyses were performed using GraphPad 7.0 software (GraphPad Software, Inc.), and P < 0:05 was considered to indicate a statistically significant difference.

Expression of lncRNA EZR-AS1 and EZR in the
Peripheral Blood. Initially, RT-qPCR was used to analyze the expression of EZR-AS1 and EZR in the peripheral blood of 35 patients with CHD and 38 control subjects. The results revealed that EZR-AS1 expression was markedly upregulated in patients with CHD, compared with the controls (Figure 1(a)). Similarly, EZR expression was also elevated in patients with CHD (Figure 1(b)).

Effects of lncRNA EZR-AS1 on Proliferation, Migration, and Apoptosis in HUVECs.
To explore the functions of EZR-AS1 in HUVECs, the cells were transfected with siRNA or overexpression pcDNA of EZR-AS1; transfection efficiency is illustrated in Figure 2(a). The results of the CCK-8 assay showed that EZR-AS1 knockdown reduced cell viability (Figure 2(b)). Additionally, si-EZR-AS1 suppressed the migration (Figures 2(c) and 2(d)) and apoptosis (Figure 2(e)) of HUVECs. Overexpression of EZR-AS1 resulted in the reverse effect.

Effects of lncRNA EZR-AS1 on the Expression Levels of
Apoptosis-Related Proteins in HUVECs. To determine whether lncRNA EZR-AS1 mediated apoptosis, Western blotting was used to detect the expression levels of apoptosis-related proteins, including PTEN, VEGF, Bax, and Bcl-2. EZR-AS1 knockdown increased the expression levels of VEGF and Bcl-2, while decreasing those of PTEN and Bax. The opposite effect was observed when EZR-AS1 was overexpressed (Figures 3(a)-3(e)). These data indicate that downregulating the expression of lncRNA EZR-AS1 inhibits apoptosis in HUVECs.

lncRNA EZR-AS1 Positively Regulates SMYD3 and EZR
Expression. The expression levels of SMYD3 and EZR were analyzed via lncRNA EZR-AS1 knockdown or overexpression. The results showed that the downregulation of EZR-AS1 decreased the expression of SMYD3 and EZR, and the opposite effect was observed when EZR-AS1 was overexpressed. These results indicate that lncRNA EZR-AS1 positively regulates the expression of SMYD3 and EZR (Figures 4(a) and 4(b)).

Overexpression of EZR Reverses the Effects of EZR-AS1
Knockdown on the Biological Properties of HUVECs. To further explore whether EZR was involved in the effects of si-EZR-AS1, HUVECs were transfected with an EZR overexpression pcDNA. RT-qPCR and Western blotting confirmed that the EZR expression level was significantly decreased in cells transfected with both si-EZR-AS1 and pcDNA-EZR (Figures 5(a) and 5(b)), compared with those transfected with pcDNA-EZR alone. Proliferation, migration, and apoptotic capacity were evaluated after transfection; the results showed that the overexpression of EZR significantly reversed the effects of si-EZR-AS1 on cell proliferation, migration, and apoptosis ( Figures 5(c)-5(g)).

Discussion
Cardiovascular diseases, particularly CHD and stroke, remain the leading global causes of death, and the lifetime risk of CHD is 67% in individuals > 55 years of age [19][20][21]. Therefore, new strategies for the treatment of CHD are urgently required. lncRNAs have been associated with the treatment of various cardiovascular diseases, including CHD [22,23]; thus, defining the functions of lncRNAs may aid in the development of novel diagnostic and therapeutic targets for CHD. Generally speaking, CHD is diagnosed when stenosis of the coronary artery reaches >50%. In the study, RT-qPCR was used to analyze the expression of EZR-AS1 in the peripheral blood of patients with severe CHD (with stenosis > 80%) and controls. The results showed that EZR-AS1 was robustly upregulated in patients with severe CHD. To the best of our knowledge, this was the first study to have evaluated the role of lncRNA EZR-AS1 in CHD. Based on these findings, the functions of lncRNA EZR-AS1 were further assessed through in vitro experimentation.
Vascular endothelial cells are important components of the vascular wall. The abnormal proliferation and migration of vascular endothelial cells are important pathophysiological processes in diseases such as CHD, hypertension, and restenosis after percutaneous coronary intervention [24][25][26]. In the present study, the role of EZR-AS1 was evaluated in HUVECs by assessing cell proliferation, migration, and apoptosis; this was achieved by transfection with si-EZR-AS1 or an overexpression pcDNA. The results revealed that the downregulation of EZR-AS1 significantly inhibited HUVECs proliferation, migration, and apoptosis; the opposite effects were observed following EZR-AS1 overexpression. These findings suggest that lncRNA EZR-AS1 influences the malignant behaviors of HUVECs, providing a basis for its therapeutic application in CHD.
EZR, encoded by Vil2, was identified as the first member of the ERM family. EZR is upregulated in a number of diseases, such as breast and cervical cancer, and overexpression of the EZR gene may enhance the metastatic capacity of tumors [27,28]. Although the function of EZR has been extensively studied, the transcriptional regulation of the EZR gene is still poorly understood. To the best of our knowledge, the role of EZR in the progression of CHD remains unclear. In the current study, EZR was revealed to be upregulated in patients with CHD, and as predicted, increased levels of EZR promoted the proliferation and migration of HUVECs. Similar to the effects of the lncRNAs BDNF-AS and NEXN-AS1 on target gene expression [29,30], knocking down EZR-AS1 decreased the expression level of EZR. Additionally, EZR overexpression reversed the effects of EZR-AS1   pcDNA-SMYD3 pcDNA-SMYD3 pcDNA-control si-EZR-AS1 si-control Control ⁎⁎ (i) Figure 6: EZR-AS1 mediates EZR expression via SMYD3. After transfected with pcDNA-SMYD3 or si-EZR-AS1+pcDNA-SMYD3, the (a, b) mRNA and (c, d) protein expression levels of SMYD3 and EZR were determined by reverse transcription-quantitative PCR and Western blotting. (e) Cell viability was detected by CCK-8 assay. (f, h) Migratory ability was detected by wound healing assay. (g, i) Apoptosis was detected by flow cytometry. Wound healing was quantified. Magnification, ×100 (migration). All results were expressed as mean ± SD. * P < 0:05 and * * P < 0:01 vs. si-control. # P < 0:05 and ## P < 0:01 vs. pcDNA-control. & P < 0:05 and && P < 0:01 vs. pcDNA-SMYD3. 9 BioMed Research International knockdown, indicating that EZR is involved in regulating the biological behaviors of HUVECs. Furthermore, siRNA suppressed the expression of EZR-AS1 while simultaneously raised that of SMYD3, which could reverse the downregulation of EZR mediated by the si-EZR-AS1, which suggested that EZR-AS1 is most likely to regulate EZR expression through SMYD3 signaling in HUVECs; more experiments such as RNA-binding protein immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) should be done in our next work to further confirm it.

Conclusion
In conclusion, and to the best of our knowledge, the present study is the first to report that lncRNA EZR-AS1 is upregulated in severe CHD and that the downregulation of EZR-AS1 inhibits the proliferation, migration, and apoptosis of HUVECs via SMYD3. These data facilitate a deeper understanding of the molecular mechanisms of lncRNA EZR-AS1 in CHD. Therefore, lncRNA EZR-AS1 may be a potential biomarker for the diagnosis and treatment of severe CHD.

Data Availability
The analyzed data sets used and/or analysed during the present study are available from the corresponding author on reasonable request.