Protection of Human Umbilical Vein Endothelial Cells against Oxidative Stress by MicroRNA-210

Oxidative stress induces endothelial cell apoptosis and promotes atherosclerosis development. MicroRNA-210 (miR-210) is linked with apoptosis in different cell types. This study aimed to investigate the role of miR-210 in human umbilical vein endothelial cells (HUVECs) under oxidative stress and to determine the underlying mechanism. HUVECs were treated with different concentrations of hydrogen peroxide (H2O2), and cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ATP assay. To evaluate the role of miR-210 in H2O2-mediated apoptosis, gain-and-loss-of-function approaches were used, and the effects on apoptosis and reactive oxygen species (ROS) level were assayed using flow cytometry. Moreover, miR-210 expression was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and expression of the following apoptosis-related genes was assessed by qRT-PCR and Western blot at the RNA and protein level, respectively: caspase-8-associated protein 2 (CASP8AP2), caspase-8, and caspase-3. The results showed that H2O2 induced apoptosis in HUVECs in a dose-dependent manner and increased miR-210 expression. Overexpression of miR-210 inhibited apoptosis and reduced ROS level in HUVECs treated with H2O2. Furthermore, miR-210 downregulated CASP8AP2 and related downstream caspases at protein level. Thus, under oxidative stress, miR-210 has a prosurvival and antiapoptotic effect on HUVECs by reducing ROS generation and downregulating the CASP8AP2 pathway.


Introduction
Atherosclerosis is the leading cause of coronary heart disease, with high mortality and morbidity rates [1]. Under oxidative stress, excessive reactive oxygen species-(ROS-) induced endothelial cell apoptosis plays a crucial role in atherosclerosis development [2]. Therefore, reduction of endothelial apoptosis is vital in atherosclerosis prevention.
The association of microRNAs (miRNAs), which are involved closely in various cell processes through posttranscriptional downregulation of their target mRNAs, with apoptosis has drawn much attention [3][4][5][6]. In particular, expression of miRNA-210 (miR-210) has been considered a general response to hypoxia [7], and miR-210 has been reported to be participated in various cellular processes, including cell cycle arrest [8], angiogenesis [6], and cell survival [5] in various cell types, including endothelial cells [6]. Besides hypoxia, miR-210 is involved in oxidative stressinduced injury. Our previous study has revealed that miR-210 protects cardiomyoblasts against apoptosis under oxidative stress [9]. However, the role of miR-210 in cell apoptosis apparently varies with cell types and stimuli [10][11][12][13]. It has been reported that miR-210 protects cardiomyocytes against apoptosis under hypoxic conditions [10] and protects stem cells against oxidative stress [11]. However, some studies identified miR-210 as a proapoptotic component in human pulmonary artery epithelial cells [12] and in a human lung cancer cell line [13]. Therefore, the role of miR-210 in apoptosis in different cell types and under different conditions needs further investigation.
Some studies have indicated that miR-210 exerts antiapoptosis action by downregulating caspase-8-associated protein 2 (CASP8AP2) in stem cells under hypoxic conditions [5]. CASP8AP2 (or FLICE-associated huge protein), a 1,962-amino acid-long protein, takes part in several cellular processes, such as mRNA processing, cell cycle, apoptosis, and transcriptional control [14]. CASP8AP2 was identified as a proapoptosis member that acts via binding the death effector domains of procaspase-8, which activates caspase-8 and its downstream caspases, including caspase-3, thereby participating in the extrinsic apoptosis pathway mediated by CD95 [15,16].
However, the effect of miR-210 on endothelial cells under oxidative stress remains unclear. In this study, we investigated the effects of miR-210 on apoptosis and the underlying mechanism in human umbilical vein endothelial cells (HUVECs) treated with hydrogen peroxide (H 2 O 2 ), which has been commonly used to induce oxidative stress in in vitro models [17]. We found that miR-210 protected HUVECs against oxidative stress by reducing ROS generation and downregulating the CASP8AP2 pathway.

Cell Culture.
HUVECs were obtained from the Department of Cardiology of The Second Hospital of Jilin University and cultured in a humidified incubator at 5% CO 2 and 37 ∘ C. The complete growth medium for this cell line consisted of the vascular cell basal medium (ATCC, USA) with the endothelial cell growth kit-BBE (ATCC, USA) and contained the following components: 2% fetal bovine serum, 0.2% bovine brain extract, 10 mmol/L L-glutamine, 1 g/mL hydrocortisone hemisuccinate, 5 ng/mL rhEGF, 50 g/mL ascorbic acid, and 0.75 units/mL heparin sulfate.

Lentivirus Infection of HUVECs.
A replication-deficient lentivirus was purchased from Sangon Biotech (Shanghai, China), encoding the human miR-210 precursor (pre-210), miR-210 inhibitor (anti-miR-210), or miR-scramble (miR-Scr, without miR-210 as a control) labeled with green fluorescent protein. 5 L of lentiviral vectors was added per well for transduction of HUVECs in 6-well plates (1 × 10 5 cells/well) for 12 h. Fresh complete growth medium was subsequently added to replace the medium. To eliminate nontransduced cells, puromycin (Sigma-Aldrich, USA) was added to the medium at a final concentration of 4 g/mL [18], and the medium was incubated for at least 72 h. The transduced cells were then stained with DAPI and observed with a fluorescence microscope. The Image J software was used to quantitate the transduction efficiency.

DAPI Staining.
After transduction with the lentivirus, cells were stained with DAPI to visualize the nucleus. Cells were cultured in a 6-well plate and fixed in 4% paraformaldehyde for 25 min at room temperature (25 ∘ C). Subsequently, cells were incubated with DAPI for 10 min and washed with PBS thrice.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Detection.
MTT detection was used to evaluate the viability of HUVECs treated with different concentrations of H 2 O 2 and the effect of miR-210 on cell viability. Cells in a 96-well plate (1 × 10 4 cells/well) were treated with different concentrations of H 2 O 2 (0, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mmol/L) for 24 h. MTT (Biosharp, China) was added to each well, and the wells were incubated for 4 h at 37 ∘ C. Subsequently, the medium was removed, and dimethyl sulfoxide (Sigma-Aldrich, USA) was added (150 L/well) to solubilize the formazan crystals. Optical density was measured at 570 nm with a Varioskan Flash microplate reader (Thermo Scientific, USA), and cell viability was calculated as a percentage of the control optical density.   (1 : 1000; Cell Signaling Technology, USA), monoclonal rabbit anti-caspase-3 (1 : 1000; Cell Signaling Technology, USA), and monoclonal mouse anti--actin (1 : 2000; Proteintech, USA) antibodies, followed by incubation with a secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody or anti-mouse IgG antibody (1 : 1000; Beyotime, China). The blots were visualized using the SuperSignal West Pico kit (Thermo Scientific, USA). The relative expression level of each protein was normalized to that of -actin.

Statistical
Analysis. Data are reported as means ± SE. Statistical analysis was performed using SPSS13.0 (IBM, USA). For all experiments, Student's -test was employed to assess statistical significance, and the threshold was set at = 0.05.

The Effect of miR-210 on HUVEC Apoptosis in Response to H 2 O 2 .
Since miR-210 expression increased in HUVECs treated with H 2 O 2 , we tested the effect of miR-210 on HUVECs apoptosis upon H 2 O 2 treatment using gain-andloss-of-function approaches. HUVECs were transduced with lentiviral vectors and observed under a fluorescence microscope (Figure 3(a)). The Image J software was used to quantitate the transduction efficiency (Figure 3(b)). The mean transduction efficiency of miR-Scr, pre-210, and anti-miR-210 was 0.88, 0.89, and 0.86, respectively. qRT-PCR showed that miR-210 expression levels were increased in the pre-210 cell line and decreased in the anti-miR-210 cell line compared with that in normal HUVECs (all < 0.05). The level of miR-210 in the control cell line transfected with miR-Scr did not differ from that in normal HUVECs (Figure 3 (Figures 6(a)-6(c)). These results indicated that the CASP8AP2 pathway was involved in HUVEC apoptosis during H 2 O 2 treatment.

Discussion
Endothelial cell apoptosis under oxidative stress plays a critical role in the initiation and progression of atherosclerosis [17]. In this study, we found that H 2 O 2 induced HUVEC apoptosis through CASP8AP2 pathway activation. In addition, H 2 O 2 stimulation upregulated the expression of miR-210, which protected HUVECs against H 2 O 2 -induced apoptosis by reducing ROS generation and affecting the CASP8AP2 pathway. We found that H 2 O 2 induced HUVEC apoptosis in a dose-dependent manner. In addition, CASP8AP2 pathwayrelated proteins were upregulated by the H 2 O 2 treatment, which further contributed to the process of apoptosis. CASP8AP2 is a member of the apoptosis signaling complex that activates caspase-8, which results in the activation of caspase-3, eventually leading to apoptosis [5,19]. In agreement, our results showed that the CASP8AP2 protein expression level increased in miR-Scr HUVECs treated with H 2 O 2 . Furthermore, we found that the cleaved caspase-8/caspase-8 and cleaved caspase-3/caspase-3 ratios increased, reflecting elevated activation of the initiator and effector caspases (caspase-8 and caspase-3, resp.) that intensified apoptosis. Therefore, activation of the CASP8AP2 pathway leads to apoptosis in HUVECs under oxidative stress.
Moreover, miR-210 was upregulated in HUVECs during H 2 O 2 stimulation. Some studies, including our previous work, showed that miR-210 upregulation had an antiapoptotic effect in the rat H9C2 cell line [9], human mesenchymal stem cells [20], and mouse myoblast C2C12 cell line [21]. However, other studies showed that miR-210 had a proapoptotic effect in pulmonary artery epithelial cells [12], human lung cancer cell line [13], and esophageal cancer cell line [22]. Of interest, previous study demonstrated that miR-210 played different roles in HCT116 and MCF7 cells at different conditions [23]. In particular, miR-210 played a proapoptotic role at normal oxygen levels and an antiapoptotic role under hypoxia [23]. Our study found that miR-210 protected HUVECs against H 2 O 2 -induced apoptosis. In line with our results, Fasanaro et al. reported that miR-210 also had a prosurvival effect on HUVECs under hypoxia [6]. Furthermore, we found a negative correlation between miR-210 level and ROS generation upon H 2 O 2 treatment. Increased ROS production is known to accelerate cell impairment. Our results indicated that miR-210 protected cells against apoptosis, which could be attributed to reduced ROS production under oxidative stress. Similar results were found in cardiomyocytes [4], mesenchymal stem cells [11], and ischemic hindlimbs [21]. However, contrasting results were obtained in colorectal cancer cells [24] and adipose-derived stem cells [25]. These differences could be due to the different cellular contexts. The underlying molecular mechanism of the reduced ROS generation could be related to the regulation of the c-Met pathway [11] and the expression of iron-sulfur cluster scaffold homolog [23].
We found that the antiapoptotic effect of miR-210 was also associated with the CASP8AP2 pathway. Overexpression of miR-210 inhibited the protein expression level of CASP8AP2 and thus decreased the cleaved caspase-8/caspase-8 ratio during H 2 O 2 treatment, while miR-210 inhibition led to the opposite effect. These results suggested that miR-210 downregulated the CASP8AP2 pathway under oxidative stress.
Although CASP8AP2 was identified as a target of miR-210 in bone marrow-derived mesenchymal stem cells under anoxia [5], Barile et al. found no association between miR-210 and CASP8AP2 in HL-1 cells [26], which indicates that the target genes of miR-210 may vary with different cell types and conditions. Moreover, numerous studies identified several other target genes of miR-210, including Efna3 [27], neuronal pentraxin 1 [28], E2f3 [29], PTPN2 [30], and ptp1b [10], suggesting that miR-210 has diverse roles in angiogenesis, DNA repair, cell cycle, migration, and apoptosis. Further studies on the association between miR-210 and other potential target genes in HUVECs under oxidative stress need to be conducted.
Of note, we found no differences in mRNA expression of CASP8AP2, caspase-8, and caspase-3 during H 2 O 2 treatment versus control cells. It is possible that alterations in mRNA expression occurred prior to the changes in protein levels.
Our results indicate that miR-210 protects HUVECs against oxidative stress in in vitro models. However, further research is needed to investigate the role of miR-210 in in vivo models and determine the underlying mechanisms.

Conclusions
We observed that miR-210 expression increased in HUVECs treated with H 2 O 2 . Moreover, miR-210 played a prosurvival and antiapoptotic role in HUVECs under oxidative stress by reducing ROS generation and downregulating the CASP8AP2 pathway. However, apoptosis-related pathways other than the CASP8AP2 pathway are known to exist. Further research on the relationship between miR-210 and these pathways is warranted. Our result may potentially lead to a new strategy for protecting against endothelial injury in atherosclerosis.