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Fangjie Chen, Hong Liu, Jingjing Wu, Yanyan Zhao, "miR-125a Suppresses TrxR1 Expression and Is Involved in H2O2-Induced Oxidative Stress in Endothelial Cells", Journal of Immunology Research, vol. 2018, Article ID 6140320, 7 pages, 2018. https://doi.org/10.1155/2018/6140320
miR-125a Suppresses TrxR1 Expression and Is Involved in H2O2-Induced Oxidative Stress in Endothelial Cells
Thioredoxin reductase (TrxR), an antioxidant enzyme dependent on nicotinamide adenine dinucleotide phosphate, plays a vital role in defense against oxidative stress. However, the role of microRNAs targeting TrxR under oxidative stress has not yet been determined. In this study, we tested the involvement of miRNA-mediated posttranscriptional regulation in H2O2-induced TrxR1 expression in endothelial cells. Dual luciferase assay combined with expression analysis confirmed that miR-125a suppressed TrxR1 expression by targeting its 3-UTR. Furthermore, H2O2 induced TrxR1 expression partly through downregulation of miR-125a. These findings indicate that miRNA-mediated posttranscriptional mechanism is involved in H2O2-induced TrxR1 expression in endothelial cells, suggesting an important role of miRNAs in the response to oxidative stress.
Growing evidence has shown that oxidative stress may be relevant to a wide range of diseases like cardiovascular disease, tumor, aging, and neurodegenerative disease [1–4]. Indeed, oxidative stress is usually caused by excessive production of reactive oxidative species (ROS) and impaired antioxidant mechanisms . In mammalian cells, the major antioxidant system includes the superoxide dismutase (SOD), glutathione peroxidase (Gpx), catalase (CAT), and thioredoxin system [6, 7]. Well-functioning antioxidant systems are essential for redox homeostasis of cells.
The thioredoxin system comprises thioredoxin (Trx), Trx reductase (TrxR), and NADPH. In this system, TrxR acts in regulating cellular oxidation reduction and protecting cells from oxidative damage by keeping Trx in a reduced state. In addition, TrxRs sustain versatile cellular functions including cell growth, apoptosis, and differentiation [8, 9]. In mammals, there are three identified TrxRs: TrxR1 in the cytoplasm, TrxR2 in mitochondria, and testis-specific isoform TrxR3. TrxR1 is present in most tissues and is denoted as the main and predominant TrxR of the three. Aberrant TrxR1 is found in the development of cardiovascular diseases. TrxR1 mRNA was significantly increased in monocytes of hypertension patients and in atherosclerotic plaques [10, 11], and the serum TrxR activity was significantly increased in coronary artery disease . These data provide empirical evidence that TrxR1 is involved in the development of cardiovascular diseases. To date, the molecular mechanism underlying the transcription regulation of TrxR1 has been well investigated. Activation of nuclear transcription factors, including Sp1, Sp3, Oct-1, and NrF2, has proved to be crucial to transactive TRXR1 . Studies have also suggested that AU-rich elements (AREs) and SECIS in the TRXR1 3-untranslated region (3-UTR) regulate its mRNA stability . However, no microRNAs (miRNAs) targeting the TRXR1 3-UTR have been reported until now.
MicroRNAs are a family of small noncoding RNAs that modulate gene expression by partially base pairing with 3-UTR of their targets . Recent evidence showing altered miRNA expression in the setting of oxidative stress suggests their involvement in oxidative stress and antioxidant defense . Computational searching with TargetScan (http://www.targetscan.org/) and PicTar (https://pictar.mdc-berlin.de/) displayed a putative miR-125a binding sequence within the 3-UTR of TrxR1 (NM_001093771). It provides the possible involvement of miRNAs in the process of TRXR1 expression. Studies have demonstrated that H2O2 is commonly used as an inducer of oxidative stress. Therefore, in this study, we identified miRNA targeting the 3-UTR of TRXR1 and elucidated its impact on TrxR1 under H2O2 treatment in endothelial cells.
2. Materials and Methods
2.1. Vector Constructs
The 3-UTR of TRXR1 was amplified by PCR and cloned into a pGL3-promotor vector (Promega) to produce the pGL3-UTR. The vector containing mutations in the miR-125a binding site of the pGL3-UTR is named as pGL3-UTR-mut. The human miR-125a precursor sequence was amplified and inserted into the pcDNA3.1(+) (Invitrogen) to generate miR-125a-expressing plasmid, pmiR125a. The control vector pcDNA3.1(+) was named pmiR-ctrl. DNA sequencing was performed to verify the orientation and authenticity of all of the inserts. The PCR primers for vector construction are listed in Table 1.
2.2. Cell Culture and Reagents
Human embryo kidney HEK293 cells and human umbilical vein endothelial cells (HUVECs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO), with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 IU/ml penicillin, at 37°C in a humidified atmosphere with 5% CO2. H2O2 treatment was carried out at concentration of 0, 0.1, 0.25, and 0.5 mmol/L for 24 h, or at 0.25 mmol/L for 0, 2, 6, 12, and 24 h, respectively. In some of the experiments, the HUVECs were stimulated by a transcription inhibitor, actinomycin D (5 μg/ml), before H2O2 treatment and harvested after a certain time.
2.3. Luciferase Reporter Assay
Cells were seeded in 24-well plates and cotransfected with 500 ng of pmiR125a or 100 nmol/L of miR-125a inhibitor (Ambion) and 200 ng of pGL3-UTR or pGL3-UTR-mut, the pRL-TK plasmid (20 ng) as the internal control. After 48 h of transfection, the Firefly and Renilla luciferase activities were determined using a luminometer (Berthold).
2.4. Detection of miR-125a and TRXR1 mRNA Expression
Total RNA extracts were prepared from treated or untreated cells using Trizol reagent (Invitrogen). For quantitative analysis of miR-125a, 2 μg of RNA was reverse-transcribed using the miRNA-specific stem-loop primer (Table 1). Real-time quantitative PCR using SYBR Green (Takara) was performed on an ABI Prism 7500 Sequence Detection System, and the expression of miR-125a was detected using the 2−ΔΔCt method with U6 as an internal control. For TrxR1 quantitative analysis, real-time PCR was applied, and GAPDH was amplified as a normalization control. The comparative Ct method was used to calculate the relative expression level.
2.5. Western Blot
Total protein was extracted from cells, and protein concentrations were determined by Bradford assay (Bio-Rad). Equal amounts of protein were separated in 10% SDS-PAGE and then transferred to a PVDF membrane (Sigma-Aldrich) at 4°C. Membranes were subsequently incubated with the anti-TrxR1 antibody (1 : 2000) (Abnova) or GAPDH antibody (1 : 5000) (Kangcheng) as the primary antibody and followed by HRP (1 : 5000) as the secondary antibody. The final detection reaction was performed with enhanced chemiluminescence detection system (Pierce, Rockford, IL) according to the manufacturer’s instructions.
2.6. Statistics Analysis
All values were expressed as mean ± SD from three independent experiments, and comparisons between quantitative variables were performed using an independent sample t-test. was considered to be statistically significant.
3.1. Identification of the miR-125a Target Site in 3-UTR of TRXR1
To find miRNAs that regulated TRXR1, we performed bioinformatics analysis using TargetScan and PicTar and found a putative miR-125a target site in TRXR1 3-UTR, which was highly conserved across species (Figures 1(a) and 1(b)). The target site was then confirmed by luciferase assay after cotransfection with pGL3-UTR (or pGL3-UTR-mut) and pmiR-125a (or pmiR-ctrl) in HEK293 cells. As Figure 1(c) shows, pmiR-125a transfection resulted in a marked descent (52%, ), but the miR-125a inhibitor gave rise to a marked ascent of luciferase activity of pGL3-UTR (23%, ). However, for the pGL3-UTR-mut, either overexpression or inhibition of miR-125a did not significantly change luciferase activity. According to these data, miR-125a might have bound to the specific sequence in the TRXR1 3UTR.
3.2. miR-125a Represses TrxR1 Protein Expression
To determine the suppression of TrxR1 expression by miR-125a, we detected both protein and mRNA levels of endogenous TrxR1 in the HUVECs after miR-125a modulation. The Western blot showed that the abundance of the TrxR1 protein significantly reduced (31%, ) when overexpressing miR-125a and increased by 47% after transfection of miR-125a inhibitor (Figure 2(a)). Moreover, the qRT-PCR result showed a nonsignificant increase of TRXR1 mRNA compared with the control (Figure 2(b)), suggesting that miR-125a did not induce TrxR1 mRNA degradation. These results suggest that miR-125a may suppress the expression of TrxR1 at the posttranscription level.
3.3. H2O2 Induces TrxR1 Expression but Downregulates miR-125a Expression in Endothelial Cells
To evaluate the impact of oxidative stress on TrxR1, we examined TrxR1 mRNA and protein in HUVECs exposed to H2O2 for different dose and time. As shown in Figures 3(a) and 3(b), both mRNA and protein levels were significantly induced by H2O2 for 2–24 h or at 0.1–0.5 mmol/ml (). When cells were exposed to 0.25 mmol/ml H2O2 for varying amounts of time, a 24 h treatment was required to reach translation peak (3.0-fold over control). Meanwhile, to determine the impact of H2O2 on miR-125a, real-time PCR was performed. MiR-125a expression was decreased by 80% () in response to the H2O2 stimulation (0.25 mmol/L) compared with untreated controls (Figure 3(c)). The results obtained from three independent experiments indicate that H2O2 upregulated TrxR1 mRNA and TrxR1 protein but downregulated miR-125a.
3.4. Posttranscriptional Regulation of TrxR1 Expression under Oxidative Stress
HUVECs were pretreated using actinomycin D (10 ug/ml) for 2 h and then exposed to H2O2 for an additional 24 h. Complete inhibition of H2O2-induced TrxR1 mRNA expression was found in the group treated with actinomycin D (Figure 4(a)). However, an evident increase in TrxR1 protein was detected in the H2O2-stimulated group after treatment with actinomycin D (Figure 4(b)). The level of TrxR1 protein in cells treated with actinomycin D and H2O2 was ~70% of the TrxR1 level in cells treated with H2O2. The preceding results suggest the involvement of a posttranscriptional mechanism in H2O2-induced TrxR1 expression in HUVECs.
3.5. Downregulation of miR-125a Is Involved in H2O2-Induced TrxR1 Expression
Given the previous results, we speculated that H2O2 could relieve the miR-125a-mediated TrxR1 suppression by miR-125a inhibition. To confirm this hypothesis, HUVECs were transfected with TRXR1 3-UTR luciferase plasmid containing the binding site of miR-125a. Cells simultaneously exposed to H2O2 for 24 h reversed the decrease of TRXR1 3UTR-associated luciferase activity compared with non-H2O2-treated control. There was no marked change of luciferase activity in H2O2-treated cells transfected with mutant and empty vector control (Figure 5(a)).
To determine whether relief of miR-125a-mediated TrxR1 translational repression was involved in H2O2-induced TrxR1 protein expression, we transfected cells with an miR-125a expression vector for 48 h and then treated cells to H2O2 for 24 h. As shown in Figures 5(b) and 5(c), overexpression of miR-125a significantly depressed H2O2-induced TrxR1 protein expression, but did not decrease TrxR1 transcription compared with H2O2-treated cells, which were also transfected with the control vector. The preceding results indicate that the relief of miR125a-mediated translational repression of TrxR1 was involved in H2O2-induced TrxR1 protein expression in HUVECs.
We confirmed that targeting of the TRXR1 3-UTR by miR-125a resulted in TRXR1 translational suppression. In addition, we found that H2O2-induced oxidative stress increased the TrxR1 expression but downregulated miR-125a expression. Moreover, H2O2-induced TrxR1 expression in HUVECs partially involved negation of miR-125a-mediated translational suppression. These results indicate that miR-125a was involved in the H2O2-induced expression of TRXR1, which may be relevant to the regulation of cell responses against oxidative stress in endothelial cells.
To date, extensive research has suggested the functions of miRNAs on oxidative stress-related genes. Eades et al. reported that miR-200a led to Keap1 mRNA degradation by targeting the 3-UTR of keap1 . Dong et al. showed that the expression of GSR and POR was suppressed by alcohol-induced miR-214 in liver cells . In addition, miRNAs may be regulated by ROS. Simone et al. revealed that a number of miRNAs including let-7b, miR-15b, and miR-21 increased under ionizing radiation, etoposide, and H2O2 in human fibroblasts . Thulasingam et al. showed that miR-21 was upregulated while miR-27a decreased under H2O2-induced stress in PC12 cells . In this work, we verified that miR-125 was directly bound to 3-UTR of the TrxR1 gene and repressed its endogenous expression, supplying another posttranscriptional regulation mechanism of TrxR1. Moreover, miR-125a expression was significantly downregulated after exposure of endothelial cells to H2O2. In addition, overexpression of miR-125a significantly depressed H2O2-induced TrxR1 protein expression. These findings suggest that miR-125a mediating the downregulation of TrxR1 plays an important role in H2O2-induced oxidative stress in endothelial cells. miR-125a was first identified in the brain tissue of mice by Northern blot in 2002 . Recently, it has been confirmed that miR-125a exerts growth regulation, lipid uptake, and vasomotor homeostasis through targeting p53, oxysterol binding protein-related Protein 9, and endothelin-1 (ET-1) genes [22–24]. Our study confirmed that TRXR1 was a newly identified target of miR-125a. To our knowledge, miRNAs are always fine-tuning posttranscriptional regulators of target mRNAs in most biological processes, including in cellular responses to redox imbalance . Therefore, we postulated that miR-125a could be a key posttranscriptional regulator in oxidative stress-mediated diseases. When assessing the effect of miR-125a in H2O2-induced oxidative stress, we found that TrxR1 was markedly increased after H2O2 treatment, consistent with the results of Furman et al. . However, mir-125a decreased significantly in H2O2-treated HUVECs. ROS modulating oxidation-sensitive signaling pathways and transcription factors is the common mechanism responsible for ROS-mediated genes. Further research should be performed to elucidate the underlying mechanism.
An imbalance between oxidative stress and the antioxidative system in endothelial cells is generally considered to be the common mechanism causing cardiovascular diseases. To keep redox-balanced conditions, cells always protect themselves from oxidative injury through activation of the antioxidant system [26, 27]. H2O2-induced TrxR1 expression results in further scavenging of ROS. Furthermore, downregulated miR-125a in the setting of oxidative stress relieves miR-125a-mediated translational repression of TrxR1, which thereby functions better in antioxidant defense.
In conclusion, miR-125a targeted TRXR1 3UTR and resulted in downregulation of endogenous TRXR1 expression in HUVECs. Moreover, miR-125a was involved in H2O2-induced oxidative stress. These results indicate that miR-125a may play a vital role in antioxidant defense via posttranscriptional regulating TRXR1 and may be a new target to regulate endothelial function.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of the article.
This research was supported by the National Natural Science Foundation of China No. 81400709.
Figure S: The effect of pmiR-125a on mature miR-125a expression and the verified target protein P53. (Supplementary Materials)
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