Inhibition of miR-383 suppresses oxidative stress and improves endothelial function by increasing sirtuin 1

Previous research has shown that suppression of miR-383 can prevent inflammation of the endothelium, as well as postpone the development of atherosclerosis. However, the role of miR-383 in endothelial cell apoptosis in diabetes remains unclear. The aim of this study was to investigate the function of miR-383 in high glucose-induced apoptosis and oxidative stress in endothelial cells. A series of experiments involving qualitative polymerase chain reaction, cell transfection, luciferase assay, assessment of cell death, detection of catalase and superoxide dismutase concentrations, detection of intracellular reactive oxygen species (ROS), and western blot analysis were performed in this study. We found that miR-383 expression was promoted, while NAD+-dependent deacetylase and sirtuin 1 (SIRT1) expressions were suppressed in the endothelium of the aorta in db/db mice as well as in human umbilical vein endothelial cells, which were treated with high glucose (HG). Increased expression of miR-383 decreased expression of SIRT1, while suppression of miR-383 promoted expression of SIRT1 in human umbilical vein endothelial cells (HUVECs). Furthermore, suppression of miR-383 following transfection with miR-383 suppressor repressed cell death and generation of ROS in HUVECs. SIRT1 knockdown by siRNA-SIRT1 reversed the suppressive effect of miR-383 inhibition on ROS production and cell apoptosis induced by HG treatment. Overall, the findings of our research suggested that suppression of miR-383 repressed oxidative stress and reinforced the activity of endothelial cells by upregulation of SIRT1 in db/db mice, and targeting miR-383 might be promising for effective treatment of diabetes.


Introduction
Diabetes elevates the risk of cardiovascular diseases (CVD), including hypertension and atherosclerosis, due to endothelial dysfunction related to oxidative stress (OS) (1)(2)(3). In the diabetic state, excessive generation of reactive oxygen species (ROS) in the walls of vessels suppresses the bioavailability of nitric oxide (NO), decreasing endothelium-dependent relaxation and leading to severe cell injury and death (4,5). Consequently, methods to attenuate OS serve as potential ways to increase the activity of endothelial cells (ECs). Sirtuin 1 (SIRT1), an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase, protects against oxidative stress and cell death (6,7). Emerging evidence indicates that micro-RNAs (miRNAs) modulate SIRT1 expression in various physiologic processes including stress resistance, apoptosis, and energy balance (8,9). miRNAs, small endogenous non-coding RNAs, have been found to be biomarkers for various diseases (10). The function of miRNAs is modulated by binding to the 3 0 -UTR of target mRNAs, resulting in degeneration of mRNA and prohibition of translation (11). Previous studies have revealed that various miRNAs regulate the development of diabetes (12,13). Downregulation of miR-15a in the retina of patients with diabetes was found to counteract inflammation and generation of vessels (14). A study on mice with diabetes showed that the expression of miR-92a increased OS and inhibited the activity of ECs by binding to heme oxygenase-1 (15). miR-383 expression has been found to be suppressed before the onset of diabetes and to modulate death of pancreatic beta cells (16). miR-383 is also able to modulate cell death in various cancers (17,18). Expression of miR-383 triggers the death of breast cancer cells by promoting their sensitivity to DNA injury (19). Nevertheless, investigation of the expression patterns of miR-383 in the endothelium of patients with diabetes is insufficient.
In this study, we examined the expression of miR-383 in ECs in response to high glucose and determined the function of miR-383 in high glucose-induced apoptosis and oxidative stress in ECs. Furthermore, the target genes mediating the function of miR-383 in ECs were identified.

EC cultivation and high glucose supplementation
Human umbilical vein endothelial cells (HUVECs) (Lonza, USA) were cultivated in EC proliferation media (CC3024; Lonza) containing antibiotics and 10% FBS. Cells from passages 4 through 8 were used in further assays. As performed in previous studies, to prepare high glucose (HG) supplemented cell cultures, HUVECs were treated with D-glucose for 48 h, followed by investigation of miR-383 expression, cell death, and generation of ROS (20).

Luciferase assay
HUVECs were co-transfected with pGL3-SIRT1, a luciferase plasmid containing SIRT1 3 0 UTR, and miR-383 or its suppressor using the Neon Transfection System (ThermoFisher). After 48 h, luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.

Assessment of cell death
HUVECs were collected and centrifuged at 1000 g for 5 min at 4°C for supernatant elution. Binding buffer was used to resuspend the pellet. FITC annexin V and propidium iodide (PI) were added prior to a 10-min incubation at room temperature. A FACScan flow cytometer (Becton, Dickinson and Company, USA) was used to examine fluorescent signals to assess cell death.

Cell counting kit-8 (CCK-8) assay
The viability of the cells was assessed using the CCK-8 assay. In brief, at the predetermined time prior to the end of treatment, 100 mL of CCK-8 solution was added to each well, and the cells were incubated at 37°C or 4 h. The absorbance values at 450 nm were measured using a multi-well spectrophotometer (Bio-Rad, USA) at 450 nm.
Detection of catalase (CAT) and superoxide dismutase (SOD1) activity CAT and SOD1 activities were measured using a catalase assay kit and SOD assay kit (Cayman Chemical Co., USA) according to the manufacturer's instructions.
The assay system consisted of 100 mM PBS (pH 7.0, 100 mL), methanol (30 mL) and sample (20 mL HUVEC from mouse aortas). The reaction was started by adding 35 mM H 2 O 2 and the reaction mixture was incubated for 20 min at room temperature. After incubation, 10 M potassium hydroxide and chromogen were added to the mixture. After further incubation for 10 min, potassium periodate was added and incubated for 5 min at room temperature before reading the absorbance at 540 nm using a plate reader (Bio-Rad). CAT and SOD1 activities were calculated using the equation obtained from the linear regression of the standard curve.

Detection of intracellular ROS
HUVECs were incubated for 30 min with 25 mmol fluorescent probe CM-H2DCFDA, then washed twice with PBS. To detect ROS inside cells, a multi-well fluorescent spectrophotometer was used at absorbance 485-530 nm. The intensity of generation of ROS in control group was manually set at 100%.

Statistical analysis
Statistical analysis was performed by SPSS Statistics version 17.0 (USA) using ANOVA and Tukey's post hoc test. The results are reported as means ± SE, with Po0.05 considered significant.

miR-383 expression was promoted in diabetic murine aortas and in ECs exposed to HG
To examine the expression of miR-383 in diabetic mouse endothelial cell, RT-qPCR of miR-383 was carried out in db/db mice and HUVECs that received HG in vitro. As presented in Figure 1, miR-383 expression was increased in the aortas of mice with diabetes compared to wild type (WT) mice. Additionally, miR-383 expression was higher in HUVECs treated with HG than in control cells. Furthermore, expression of SIRT1 was suppressed in db/db mice and HUVECs treated with HG compared to controls. These findings indicated that increased expression of miR-383 may be related to the malfunction of ECs under diabetic conditions.

miR-383 targeted SIRT1 in ECs
The miRanda database (www.microrna.org) was used to filter and identify the putative target genes of miR-383. Bioinformatics analysis predicted that SIRT1 may serve as a target of miR-383. We found that increased expression of miR-383 suppressed the expression of SIRT1 in HUVECs and suppression of miR-383 promoted its expression (Figure 2A and B). Expression of SIRT1 was likely regulated by miR-383 binding directly to the 3 0 UTR and prohibiting translation. Suppression of miR-383 enhanced the expression of the SIRT1 protein in HUVECs ( Figure 2C and D).

Suppression of miR-383 decreased OS in ECs
It has been shown that increased OS contributes to the dysfunction of ECs under diabetic conditions (3). To investigate the influence of miR-383 on OS, we carried out loss of function assays by transfecting HUVECs with miR-383 suppressor or NC. Staining with CM-H2DCFDA fluorescent probe showed that ROS concentration increased in the HUVECs transfected with NC ( Figure 3A and B). miR-383 suppression significantly repressed the generation of ROS in HUVECs. Furthermore, miR-383 suppression elevated CAT and SOD1 activity, as effective endogenous antioxidant, compared to the NC ( Figure 3C and D). In addition, miR-383 suppression increased the activity of CAT and SOD1 in the aortas of db/db mice ( Figure 3E and F). These findings proved that suppression of miR-383 represses excessive ROS generation in ECs under diabetic conditions.

Suppression of miR-383 reduced cell death of ECs
OS stimulation triggered by HG is an essential mechanism in ECs death (22). Thus, we examined the influence of miR-383 suppression on the death of HUVECs. Supplementation of the cells with HG increased the quantity of dead cells, which was counteracted by suppression of miR-383 ( Figure 4A-C). Thus, targeting miR-383 could promote the viability of EC cells exposed to HG.

SIRT1 knockdown counteracted the influence of miR-383 suppression on ROS generation and ECs death
To examine if SIRT1 regulated the influence of miR-383 suppression on the attenuation of OS and promotion of EC viability, siRNA was used to knock down expression of SIRT1 in HUVECs ( Figure 5A and B). The influence of miR-383 suppression on the ROS generation by HUVECs following exposure to HG was counteracted by knockdown of SIRT1. Knockdown of SIRT1 repressed the reinforcement of CAT and SOD1 that is usually triggered by suppression of miR-383. Furthermore, the inhibitory influence of miR-383 suppression on HG-triggered death of HUVECs was counteracted by knockdown of SIRT1 ( Figure 5C-F). These results showed that suppression of ROS generation and death of ECs by miR-383 can be modulated by knockdown of SIRT1.
Discussion miRNAs are essential contributors to the modulation of CVD by targeting various genes (23,24). miR-383 is involved in various cellular processes related to the development of various diseases, such as generation of malignancies and cerebral ischemia (25)(26)(27). Our study found an increased expression of miR-383 in murine aortic ECs from db/db mice that were treated with HG. It was revealed that increased expression of miR-383 in diabetic murine ECs reinforced OS, and suppression of miR-383 impaired cell death and normalized ROS generation in ECs under diabetic conditions. miR-383 targeted SIRT1, whose upregulation promoted endothelial activity by suppressing miR-383 expression in db/db mice. These findings emphasized the promising influence of miR-383 on regulation of malfunctioning vessels in patients suffering from diabetes. Targeting of miR-383 may be a way to treat vessel pathologies in diabetes.
Excessive ROS generation contributes to EC malfunction in diabetes (28). Previous studies have shown that ROS generation partly contributes to HG-triggered cell death of ECs (29,30). OS normalization, resulting from use of antioxidants, reduces damage to vessels in patients with diabetes (31,32). We found that suppression of miR-383 reduced ROS generation, elevated the levels SOD1/CAT, and repressed ECs death that were supplemented with HG. This indicated that endothelial activity was promoted through miR-383 suppression in ECs by prohibition of ROS under diabetic conditions.
Our study revealed the target gene that could participate in preserving the influence of miR-383 suppression on ECs in patients with diabetes. We screened for enzyme expression related to OS in HUVECs transfected with miR-383 suppressor and found promotion of SIRT1 translation. We confirmed that suppression of Although previous research has explored the etiology of SIRT1 modulation in ECs, it has not been well studied in diabetes (33,34). In the present study, we found that SIRT1 was downregulated in HUVECs treated with HG. Downregulation of SIRT1 was also seen in ECs from the aortas of db/db mice. Our research has shown that SIRT1 suppressed miR-383 expression in diabetic murine ECs to preserve their influence on vessels.
In summary, our study provided evidence that targeting miR-383/SIRT1 axis can be an alternative therapeutic strategy for ameliorating diabetic vascular degeneration.