db mice through inhibiting the expression of FoxO1

Accepted Manuscript MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1 Guangwei Yang, Yuanbo Wu, Shandong Ye PII:

S0006-291X(17)30374-1

DOI:

10.1016/j.bbrc.2017.02.083

Reference:

YBBRC 37331

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 14 February 2017 Accepted Date: 16 February 2017

Please cite this article as: G. Yang, Y. Wu, S. Ye, MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.02.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1 Guangwei Yang1#, Yuanbo Wu2#, Shandong Ye1* 1

Department of Endocrinology, 2 Department of Neurology

China

*

These authors contributed equally to the article.

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#

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Running title: MiR-181c inhibits FoxO1 expression in DM

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Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui 230001,

Corresponding author: Shandong Ye

Mailing address: Department of Endocrinology, Affiliated Anhui Provincial Hospital,

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Anhui Medical University, Hefei, Anhui 230001, China Address: No.17 Lujiang Road, Hefei, Anhui 230001, China

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E-mail:

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Tel: 86+0551-62283346

ACCEPTED MANUSCRIPT ABSTRACT

Endothelial dysfunction played an important role in the progression of diabetes mellitus (DM). miR-181c has been implicated in many diseases, including DM.

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However, the molecular mechanisms of miR-181c regulate this process remained poorly understood. Healthy ICR mice were divided into control group (n=10) and db/db DM group (n=10). The expression of miR-181c and FoxO1 were both investigated in diabetic db/db mice or high glucose-induced endothelial cells (MAECs

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and END-D). Here we found that down-regulation of miR-181c and the activation of FoxO1/iNOS were observed in mice and endothelial cells. Furthermore, we verified

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that miR-181c directly targeted and inhibited FoxO1 gene expression by targeting its 3’-UTR through luciferase reporter assay. Knockdown of FoxO1 reversed the up-regulation of iNOS, nitrotyrosine and the down-regulation of p-eNOSSer1177/eNOS in high glucose (30 mM)-induced MAECs cells. In addition, over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while

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this effect could be attenuated by pcDNA-FoxO1 in MAECs. These results shown that miR-181c attenuated nitration stress through regulating FoxO1 expression and affecting endothelial cell function, which offering a new target for the development of

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preventive or therapeutic agents against DM.

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Keywords: MiR-181c; nitration stress; endothelial cell; diabetes mellitus; FoxO1

ACCEPTED MANUSCRIPT INTRODUCTION Diabetes mellitus (DM) with an increasing prevalence has become one of the most common metabolic diseases [1]. Endothelial dysfunction was as the earliest critical pathological event and an initial pivotal factor in the occurrence and

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pathogenesis of DM [2]. Inflammatory activation, reduced vasodilator response and increased plasma levels of endothelial products was included [3] and it was characterized by decreased nitric oxide (NO) bioavailability [4]. Previous studies presented that impaired endothelium-dependent vasodilatation has been occurred in

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patients with DM [5]. However, valid therapeutic options were quite restricted and identification of potential approach targets was urgently needed.

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Forkhead box O1 (FoxO1), as a vital transcription factor, which played an important role in regulating cellular metabolism, oxidative stress response and cell death [6]. FoxO1 might associate with pancreatic β-cell defect in DM disease [2]. Up-regulation of FoxO1 involved in DM caused metabolic disorder and cell death, FoxO1 also promoted iNOS expression and mediated oxidative stress DM [7]. In

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addition, FoxO1 couples metabolic activity and growth state in the vascular endothelium, which acted as a gatekeeper of endothelial quiescence [8]. Activation of FoxO1 by insulin resistance could promote cardiac dysfunction and β-myosin heavy

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chain gene expression [9]. However, the underlying molecular mechanism regulating the expression of FoxO1 in DM has not been fully identified. MicroRNAs (miRNAs, ~22 nt) regulated gene expression in a sequence-specific

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manner [3]. Kinds of miRNAs were participated in various biological processes and have become modulators for diseases [10-12]. Up to now, their function in DM begun to be understood. Serum miR-196 and miR-200 could be used as additional biomarker in pancreatic ductal adenocarcinoma patients with DM [13]. The expression levels of circulating miR-126 were decrease in peripheral blood of type 2 diabetes patients [14]. Moreover, miR-182 was a negative regulator of osteoblast proliferation and differentiation through targeting FoxO1 [15]. In prostate cancer, miR-96 expression inversely corrected to FoxO1 expression, up-regulation of miR-96 could enhance cellular proliferation and cancer progression [16]. Nevertheless, how miR-181c

ACCEPTED MANUSCRIPT regulated the expression of FoxO1 in DM process remained unclear, and the core mechanism of endothelial cell injury in DM was still to be determined. Therefore, in the current study, miR-181c expression and its correction with FoxO1 level in diabetic db/db mice or high glucose-induced endothelial cell were

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both investigated. Further analysis was conducted to verify the function of miR-181c on nitration stress, involving an underlying mechanism regulated by the expression of

1. Material and methods 1.1 Animals and experimental design

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FoxO1, which revealed the promising targeted role of miR-181c in DM progression.

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The animal protocols were approved by the Research Ethics Committee of Affiliated Anhui Provincial Hospital, Anhui Medical University. All operations were finished under the condition of urethane anesthesia, and all the work was performed to minimize pain.

20 healthy male ICR mice that were 8 weeks old were purchased from the

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Animal Center of Anhui Medical University (Hefei, China), and were chosen for the following experiments. Before the beginning of the study, these animals were housed and kept in air conditioned room environment for at least 1 week. Then mice were randomly divided into two groups: control group (WT), model group (db/db) with

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streptozotocin (STZ, 4×10-2 mg/g/d) [17] induced diabetes. The corresponding control mice were injected with the buffer alone. Fasting blood glucose (FG) levels higher

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than 11.8 mmol/L were determined to be successful DM model rats [17]. In addition, all these mice were provided with water and standard rodent diet ad libitum. The above-mentioned procedures lasted for 7 weeks and animals were used to diverse protocols.

1.2 Cell line and culture conditions Mouse aortic endothelial cells (MAECs) were isolated from db/db diabetic mice and the control group. Mouse endothelial cell lines END-D were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The above-mentioned cells were maintained in RPMI 1640 medium provided with 10%

ACCEPTED MANUSCRIPT fetal bovine serum, 100 units/ml penicillin and 100 µg/ml of streptomycin. All the cells were kept in humidified air at 37 °C with 5% CO2. 1.3 RNA extraction and qRT-PCR Total RNAs were isolated from tissues or cultured cells using TRIZOL reagent

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(Invitrogen, UA, USA) following the manufacturer's instructions. The same amount of total RNA (2 µg) was subjected to reverse transcription with M-MLV reverse transcriptase (Promega, Madison, WI). qRT-PCR was performed using an ABI 7300 Fast RT-PCR system with SYBR® Green Master Mix. U6 and GAPDH were used as

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endogenous controls, respectively. The relative expression level was calculated by using the 2-∆∆CT method. Statistical analysis was performed on the fold-change.

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1.4 Western blotting

Protein lysates of cells or tissues were separated by 10% SDS-poly acrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, and incubated with specific antibodies against FoxO1, iNOS, p-eNOS, eNOS. All the primary antibodies were obtained from Cell Signaling Technology, Inc (CST). GAPDH served

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as a loading control. Then the membrane loaded with proteins was incubated with a peroxidase-linked secondary antibody after washed with TBST. After antibody incubation, protein band was visualized and quantified by SmartChemi (Beijing Sage

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Creation Science Co, China).

1.5 MiR-181c mimic and inhibitor Cells were treated with miR-181c mimic or miR-545 inhibitor (Ambion Pre-miR

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miRNA Precursors, Life Technologies) using Oligofectamine (Life Technologies) according to the manufacturer’s instructions with corresponding controls. Further analysis of the samples was performed at 48 h post-transfection. 1.6 Luciferase reporter gene assay Bioinformatic analysis of miR-181c target sites was performed using TargetScan Human Release 6.2 (http://www.targetscan.org/). The 3’-UTR of FoxO1 was cloned into psiCHECK-2 vector and luciferase reporter plasmid psiCHECK-FoxO1-3’-UTR was generated. Plasmid DNA and miR-181c mimic or inhibitor or the corresponding controls were co-transfected into END-D cells by using Lipofectamine 2000

ACCEPTED MANUSCRIPT Transfection Reagent. The relative luciferase activities were quantified on 48 h after transfection using Dual Glo Luciferase Assay System (Promega, WI, USA). 1.7 Cell transfection The FoxO1 siRNAs and scrambled negative control siRNA or pcDNA-FoxO1

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and pcDNA plasmid were obtained from Invitrogen (Invitrogen, CA, USA). The si-FoxO1, pcDNA-FoxO1 and the respective control was transfected into MAECs through the use of Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, cells were harvested for qRT-PCR

1.8 Injection of miRNAs to diabetic db/db mice

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analyses or western blotting.

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For the injection of miR-181c mimic or pre-NC to diabetic db/db mice study, a total 20 db/db mice were used and randomly divided into 2 groups: pre-NC group and miR-181c mimic group. Animals were anesthetized with ketamine and xylazine, miR-181c mimic or pre-NC was injected into the thoracic aorta. All animal experiments with db/db mice were performed strictly in accordance with a protocol

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approved by Anhui Medical University.

1.9 Quantification of nitrotyrosine content The nitrotyrosine content in mice aortic and MAECs cells was detected with an

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ELISA kit following the manufacturer's instructions. The results of nitrotyrosine content were presented as pmol/mg protein, and total aorta proteins were measured

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using a BCA assay kit (Beyotime Institute of Biotechnology, Shanghai). 1.10 Statistical analysis Experimental values were expressed as means ± standard deviation. All

statistical tests were performed using Graphrad Prism software version 5.0. Comparisons between groups were analyzed using Student’s t test or ANOVA, and the Student-Newman-Keuls method was used to estimate the level of significance. Probabilities of 0.05 or less were considered to be statistically significant. All results were reproduced in at least three independent experiments.

2. RESULTS

ACCEPTED MANUSCRIPT 2.1 MiR-181c was down-regulated while FoxO1 was up-regulated in db/db mice and endothelial cells treated with high glucose Primary MAECs were isolated from normal mice and db/db model group, and the change of miR-181c and FoxO1 expression in the supernatant was analyzed.

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Firstly, endothelium-dependent vasodilatation of thoracic aorta (characterized by the diastolic responsiveness of ACh) significantly decreased in diabetic db/db mice when compared to the controls (Figure 1A); while there was no a significant difference in the relaxation response of sodium nitroprusside (Figure 1B). Moreover, miR-181c

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expression in treated diabetic mice decreased approximately 50% compared with that from control mice (Figure 1C). The mRNA and protein level of FoxO1 gene was

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obviously up-regulated in model group as compared with those of controls (Figure 1D).

To further reveal the role of miR-181c and FoxO1 on mouse endothelial cells in vitro, primary isolated MAECs and mouse endothelial cell line END-D were selected and induced with high glucose at the concentration of 30 mM for 24 h. The relative

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miR-181c mRNA expression was significantly reduced in high glucose treated groups (Figure 1E). However, FoxO1 gene and protein levels were obviously increased in endothelial cells when compared with the corresponding controls (Figure 1F). Taken

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together, these results indicated that miR-181c and FoxO1 may play important roles in

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the progression of diabetic.

2.2 MiR-181c directly targeted FoxO1 To detect the effect of miR-181c on the regulation of FoxO1, the underlying seed

sequence of FoxO1 3’-UTR was cloned into luciferase reporter constructs. END-D endothelial cells treated with miR-181c inhibitor showed significant up-regulation of luciferase activity about 2 fold compared to that of negative cells treated with pre-NC. Moreover, the mRNA and protein level of FoxO1 were also significantly increased in miR-181c silencing END-D cells (Figure 2A). In contrast, the mRNA and protein level of FoxO1, along with the relative luciferase activity were all significantly down-regulated in smooth muscle cells treated with miR-181c mimic (Figure 2B).

ACCEPTED MANUSCRIPT Therefore, we can concluded that FoxO1 could act as a directly target of miR-181c.

2.3 MiR-181c mimic reversed the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-FoxO1 in MAECs

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Given FoxO1 expression was obviously up-regulated under high glucose, and si-FoxO1 was constructed into MAECs, aiming to investigate the function of FoxO1 silencing on nitration stress. As shown in Figure 3A, high glucose-induced iNOS up-regulation was strongly reversed by si-FoxO1. The quantitative histogram was

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shown in Figure 3B. In addition, p-eNOSSer1177/eNOS ratio was decreased in high glucose treated group, while this decrease can be counteracted by further treatment

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with si-FoxO1 (Figure 3C). Further analysis for nitrotyrosine production presented that high glucose-caused nitrotyrosine content were obviously attenuated in FoxO1 intervened MAECs (Figure 3D).

To detect the role and function of pcDNA-FoxO1 on the nitration stress mediated by miR-181c in MAECs, cells treatment with high glucose were transfected with

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miR-181c mimic or pre-NC, pcDNA-FoxO1 or pcDNA. After high glucose induction, the relative iNOS protein level (Figure 3F) and nitrotyrosine content (Figure 3H) were both significantly increased, while the ratio of p-eNOSSer1177/eNOS (Figure 3G)

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was significantly decreased. Moreover, cells transfected with miR-181c mimic reversed the above mentioned changes induced by high glucose, and those effects could be eliminated by FoxO1 over-expression. Western blotting analysis was also

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consistent with the above results, including iNOS, p-eNOS and eNOS proteins (Figure 3E). Therefore, we concluded that miR-181c restrained nitration stress of endothelial cells with high glucose treatment through inhibiting FoxO1 expression.

2.4 MiR-181c over-expression decreased nitration stress in diabetic db/db mice To further verify the function of miR-181c on the nitration stress, diabetic db/db mice were selected and injected with miR-181c mimic or pre-NC. As demonstrated in Figure 4A, the endothelium dependent vasodilatation function was significantly improved in mice treated with miR-181c mimic, as compared with the corresponding

ACCEPTED MANUSCRIPT control (pre-NC). Furthermore, the relative iNOS protein expression and nitrotyrosine content in miR-181c over-expression of db/db mice were both less than those of the control group (Figure 4B and 4C). Conversely, p-eNOSSer1177/eNOS proportion was

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markedly up-regulated in mice transfected with miR-181c mimic (Figure 4B).

3. DISCUSSION

The present study provided first evidence that up-regulation of miR-181c had protective effects on endothelia in DM, partially through restraining nitration stress

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and inhibiting the expression of FoxO1, which may shed a novel light on better mechanism underlying of diabetes.

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Cardiovascular diseases were often simultaneously occurred in patients with DM, which occupied a large proportion in morbidity and mortality around the world [18]. Moreover, hyperglycemic episodes, hyperlipidemia and insulin resistance were tightly bound to the development and progression of cardiovascular metabolic diseases [19]. In general, the metabolism disorder always cause endothelial dysfunction and induce

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diverse cardiovascular disorders [20]. These findings in our study were consistent with previous reports that STZ-induced diabetes prompted endothelial dysfunction mainly manifested as significant decreased endothelium-dependent vasodilatation of

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thoracic aorta and the production of eNOS [21]. In aortic rings pre-contracted with phenylephrine, ACh relaxation was impaired in diabetic group compared to those from normal rats [22]. Endothelium-dependent relaxation induced by ACh was

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attenuated in carotid arteries from STZ-induced diabetic rats, circulating microparticles from diabetic rats could impair endothelial function and regulate eNOS expression [23]. Thus, exploring the effective therapeutic targets to improving the metabolic disturbance was urgently desired. The expression of eNOS factor or eNOS phosphorylation in the endothelium has been demonstrated to act an important role in vasorelaxation [24]. The absence of eNOS or reduced eNOS viability may induce endothelial dysfunction in most blood vessels [25]. Various specific sites on the phosphorylation eNOS protein could mediate the activity of eNOS, and the most extensively researched were the

ACCEPTED MANUSCRIPT phosphorylation of Ser1177, which was a positive regulatory site of eNOS [26]. It has been indicated that Ach can activate eNOS phosphorylation on Ser1177 [27]. Similarly, in the current study, the expression of p-eNOSSer1177 in high glucose induced endothelial cells was obviously decreased. The findings were consistent with

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others’ results. Treatment with STZ led to a significant decreased eNOS phosphorylation on the activator (Ser 1177) site [27]. The expression of the GCH-I and eNOS activating Ser1177 phosphorylation was decreased by STZ, and therapy with telmisartan prevented eNOS uncoupling in STZ-induced diabetes [28]. However,

nitration stress required further investigation.

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the mechanism by which diabetes act on the reduction in eNOS expression and

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Recently, the forkhead protein FoxO1 was activated through high glucose and oxidative stress in vascular endothelial cells. Additionally, Foxo1 promoted inducible NOS-dependent NO-peroxynitrite generation, which led in turn to LDL oxidation and eNOS dysfunction [29]. According to this study, FoxO1 mRNA level up-regulated in diabetic db/db mice and aortic endothelial cells treated with high glucose. Moreover,

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FoxO1 silencing reversed the enhanced iNOS expression induced by high glucose in MAECs. Our research finding were in accordance with Lu’s results that cutaneous FoxO1 levels increased in diabetic mice and treatment with genistein suppressed it in

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a dose-dependent manner, which mirrored the increase in ac-FoxO1 [7]. Inhibiting FoxO1-iNOS-CD36 signaling reduced cardiac lipid accumulation and nitration stress, along with ameliorated the cardiovascular complications related with diabetes [30].

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Interpretation of FoxO1/iNOS axis may contribute to the delayed wound healing in type 1 DM [31]. All these mentioned documents presented that FoxO1 played an important role in the process of DM, while further studies were needed to explore the core mechanism involved in the regulation of FoxO1. The regulation of various miRNAs was well-studies in cancers, as well as in the endothelium in response to a diabetic-like environment. MiR-15a was down-regulated in plasma of DM patients [32] and in β-cells exposed to high glucose (33 mM) for long periods [33]. MiR-181c was reduced in a diabetic-like environment and up-regulated after the addition of calcitriol [3]. In a similar manner, miR-181c was

ACCEPTED MANUSCRIPT also found to be decreased in db/db mice and endothelium cells induced with high glucose. Moreover, it has been reported that miR-181c regulated cancer stem cell proliferation and cell cycle [34]. In hypertrophic scars fibroblasts, miR-181c was abnormally expressed and targeted urokinase type plasminogen activator (uPA) to

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affect collagen type I generation [35]. Nevertheless, in the present research, we demonstrated for the first time that miR-181c impeded nitration stress of endothelial cells through inhibiting FoxO1 expression, although mounting evidence have confirmed the regulation effect between miRNAs and FoxO1 in osteoblast

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proliferation and differentiation [15], prostate cancer cell apoptosis [36], pancreatic ductal adenocarcinoma growth [37], non-small cell lung cancer cells viability [38] and

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so on.

In conclusion, the current study demonstrated that miR-181c had ameliorated the nitration stress in diabetes through suppressing the expression of FoxO1. These results could potentially provide a mechanistic basis that therapeutic interventions inhibiting

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FoxO1 expression in the diabetic patient may further improve patient outcomes.

CONFLICT OF INTEREST

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The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1 MiR-181c was down-regulated while FoxO1 was up-regulated in db/db mice and endothelial cells treated with high glucose. Two mouse endothelial cells (MAECs and END-D) were exposed to high glucose (30 mM) for 24 h. (A)

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Endothelium-dependent vasodilatation of thoracic aorta (characterized by the diastolic responsiveness of ACh). (B) Relaxation response of sodium nitroprusside. (C, E) Relative miR-181c expression was detected by qRT-PCR. (D, F) Relative FoxO1 mRNA and protein level was detected by qRT-PCR and Western blotting, respectively.

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U6 was used as the endogenous control of miR-181c, and GAPDH was used as the internal reference of FoxO1 at gene and protein level. **P<0.01 compared with the

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corresponding control group.

Figure 2 Identification of the relationship between miR-181c and FoxO1. (A) With the knock-down of miR-181c, the relative luciferase activity and FoxO1

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expression were significantly elevated in END-D endothelial cells. (B) With the artificial over-expression of miR-181c, the relative luciferase activity and FoxO1 expression were dramatically decreased in smooth muscle cells. GAPDH was used as

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pre-NC group.

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the internal reference of FoxO1 at gene and protein level. **P<0.01 compared with the

Figure 3 Over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-FoxO1 in MAECs. (A, E) The expression of iNOS, p-eNOS, e-NOS detected by Western blotting method, and GAPDH was acted as the loading control. (B, F) Relative iNOS protein level was assessed by ELISA. (C, G) The ratio between p-eNOSSer1177 and eNOS. (D, H) Nitrotyrosine content (pmol/mg protein) was determined by ELISA kit. From Figure A-D, **P<0.01 compared with control group; ##

P<0.01 compared with high glucose (30 mM) + si-NC group. From Figure E-H,

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P<0.01 compared with control group;

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P<0.01 compared with high glucose (30

mM) + pre-NC group; &&P<0.01 compared with high glucose (30 mM) + miR-181c +

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pcDNA group.

Figure 4 MiR-181c over-expression decreased nitration stress in diabetic db/db mice. Diabetic db/db mice were injected with miR-181c mimic or pre-NC, and then detected (A) Endothelium dependent vasodilatation function. (B) Relative iNOS

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protein level and p-eNOSSer1177/eNOS ratio. (C) Nitrotyrosine expression (pmol/mg

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protein). **P<0.01 compared with control (pre-NC) group.

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