miR-450a-5p eliminats MGO-induced insulin resistance via targeting CREB CURRENT STATUS: POSTED

Background miR-450a-5p was involved in fat formation, but its role in insulin resistance remains unclear. This study further investigated the effects of miR-450a-5p in endothelial cells, with the aim of finding a potential target for diabetes mellitus. Human umbilical vein endothelial cells (HUVECs) were severally treated with low-glucose, high-glucose, methylglyoxal (MGO), and insulin only or plus MGO. miR-450a-5p was up-regulated or down-regulated in treated HUVECs. miR-450a-5p expression in cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity was determined through MTT experiments. Transwell assay and oil red O staining were used for the detection of cell invasion and fat formation. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by double-luciferase reporter analysis, and its influence in eNOS/AKT pathway was estimated.


Abstract
Background miR-450a-5p was involved in fat formation, but its role in insulin resistance remains unclear. This study further investigated the effects of miR-450a-5p in endothelial cells, with the aim of finding a potential target for diabetes mellitus.

Methods
Human umbilical vein endothelial cells (HUVECs) were severally treated with low-glucose, highglucose, methylglyoxal (MGO), and insulin only or plus MGO. miR-450a-5p was up-regulated or downregulated in treated HUVECs. miR-450a-5p expression in cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity was determined through MTT experiments. Transwell assay and oil red O staining were used for the detection of cell invasion and fat formation. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by doubleluciferase reporter analysis, and its influence in eNOS/AKT pathway was estimated.
Results miR-450a-5p decreased obviously in endothelial cells with high-glucose and MGO. Through in vitro cell experiments, we knew that MGO could not only intensify the activity of endothelial cells, but also accelerate cell invasion and fat accumulation, which could be reversed by up-regulated miR-450a-5p.
Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin, which were eliminated by up-regulated miR-450a-5p. Furthermore, CREB was the target gene of miR-450a-5p that had an activation effect on the eNOS/AKT pathway.

Conclusions
Up-regulated miR-450a-5p eliminated MGO-induced insulin resistance via targeting CREB, which might be a potential target to improve insulin resistance and benefit patients with related diseases.

Background
Diabetes mellitus (DM), a common metabolic disorder, is featured by a chronically raised level of blood glucose caused by insulin dysfunction [1]. Methylglyoxal (MGO) plays a pivotal role in the occurrence and development of DM, which is a highly active dicarbonyl compound and a ubiquitous product of cell metabolism [2]. Concretely, MGO is an inherent by-product of glycolysis with cell permeability, the aggregation of which is harmful because it is the most active compound to induce glycosylation products in cells [3]. As a result, MGO will induce insulin resistance that further aggravates pathoglycemia and dyslipidemia, thus promoting the progress of the disease [4]. In addition, MGO can easily react with proteins, fats and nucleic acids to form advanced glycation end products, leading to the occurrence of cataract, retinopathy, kidney disease, vascular disease and other diabetic complications [5][6][7][8]. In recent years, although several MGO scavengers have been developed, there is no clear clinical plan for the treatment of insulin resistance induced by MGO [9]. Therefore, finding a new therapeutic target to relieve MGO-induced insulin resistance may have a positive significance in the cure of DM.
MicroRNAs (miRs) are a class of single-stranded non-coding RNAs with length of approximately 18-23 nucleotides, which exert post-transcriptional modification via binding to the 3'untranslated region of target gene, so as to affect the stability and translation of mRNA. It is estimated that miRNAs account for about 1% of the genome of nuclear organisms, and the regulatory range of their target genes covered 30% of all genes [10]. A recent study has found that miRNAs played an important role in the pathogenesis of DM by regulating the synthesis, secretion and signal transduction of insulin [11]. For example, Li X [12] indicated that miR-375 regulated the expressions of insulin secretion-related genes to control the progression of DM. In addition, some scholars have found that miRNAs also exerted a vital function in insulin signaling pathway and insulin resistance, such as miR-143, miR-145, miR-29, etc. In related fields, miR-450a-5p can secrete various factors to regulate fat formation through paracrine signals [13]. Correlatively, previous animal studies have shown that the formation of fat may be associated with the onset of DM [14]. Nevertheless, the role and mechanism of miR-450a-5p in insulin resistance of endothelial cells remains unclear.
Herein, we explored the relationship between miR-450a-5p and MGO, and further analyzed the effects and mechanisms of miR-450a-5p on MGO-induced human umbilical vein endothelial cells (HUVECs), including cell activity, invasion, fat formation and insulin resistance. The purpose of this study was to further analyze the action mechanism of miR-450a-5p in the insulin resistance induced by MGO, so as to provide a potential target for related therapies, thus delaying the progression of DM and improving the prognosis of patients.

qRT-PCR assay
The expression levels of relevant mRNAs in HUVECs were measure by qRT-PCR assays. Concretely, total RNA was separated from HUVECs using TRIzol reagent (Invitrogen, Carlsbad, California, USA).
NanoDrop-2000c spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) was adopted to assess the quality of RNA, while the integrity was determined by 1% agarose modified gel electrophoresis. Then, 1 µg of separated RNA was reverse transcribed into cDNA with the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan) according to the manufacturer's instructions.
qRT-PCR assay was conducted with the ABI Prism 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA) following the reaction conditions: total for 30 cycles of hot start for 1 min at 94°C, denaturation for 1 min at 94°C, annealing for 45 s at 50°C, and elongation for 2 min at 72°C, then final elongation for 8 min at 72°C. The sequences of primers were shpwed in Table 1 and synthesized by Gene Pharma (Shanghai, China). Using GAPDH or U6 as internal reference, and the data were evaluated with the comparative 2 -ΔΔCt method [15].

Statistical analysis
Statistical Package of the Social Sciences 20.0 software (SPSS, Inc., Chicago, USA) was used for data analysis. The measurement data were presented as mean ± standard deviation (SD). The comparison between groups was performed by Student's t-test or one-way analysis of variance (ANOVA). All experiments were repeated in triplicate. P <0.05 was considered as statistically significant.

Results
Over-expressed miR-450a-5p inhibited viability, migration, and lipid formation of MGO-induced HUVECs Based on qRT-PCR analysis, the expression level of miR-450a-5p in HUVECs was observably decreasing after 72 h of high-glucose treatment (P<0.001), while low-glucose had no obvious effect on miR-450a-5p expression ( Figure 1A). Moreover, the addition of MGO clearly inhibited the expression of miR-450a-5 in HUVECs, which could be reversed by AG (P<0.001; Figure 1B, C). In order to further investigate the effects of miR-450a-5p and MGO on the cells, transfection was performed in the MGO-induced cells. The results revealed that miR-450a-5p was obviously upregulated by mimic and down-regulated via inhibitor (P<0.001; Figure 2A), which means the transfection was successful. Interestingly, MTT results showed that over-expressed miR-450a-5p prominently restrained the viability of MGO-induced HUVECs after transfection for 48 and 72 h (P<0.001; Figure 2B), while down-regulated miR-450a-5p had no significant effect on cell viability. In Transwell assay, microscopic and quantitative analysis indicated that over-expressed miR-450a-5p observably suppressed the migration of MGO-induced HUVECs (P<0.001; Figure 2C, D), whereas down-regulated miR-450a-5p had no significant effect on cell migration. In the oil red O staining, lipid formation showed an orange appearance. Experimental images viewed that over-expressed miR-450a-5p significantly reduced the lipid accumulation of MGO-induced HUVECs ( Figure 2D), while the lipid accumulation of cells was still high after down-regulating miR-450a-5p.
Over-expressed miR-450a-5p reduced the insulin resistance of MGO-induced HUVECs by targeting CREB WB analysis suggested that insulin promoted the phosphorylation of eNOS and AKT, which could be reversed by MGO, and the addition of miR-450a-5p relieved the effects of MGO on eNOS/AKT pathway (P<0.001; Figure 3A-C). Analogously, Griess reaction displayed that MGO inhibited the insulin-induced NO release, which could be relieved by up-regulated miR-450a-5p (P<0.001; Figure 3D). For in-depth exploration, Targetscan7.2 forecasted the position 108-114 of CREB1 3'UTR was the binding site of miR-450a-5p ( Figure 4A). Double-luciferase reporter analysis indicated that the luciferase activity of HUVECs was observably depressed after co-transfection with miR-450a-5p and CREB-WT, which verified that CREB was the target gene of miR-450a-5p (P<0.001; Figure 4B). Moreover, MGO promoted the expression of CREB in HUVECs, whereas miR-450a-5p had the completely opposite effect (P<0.001; Figure 4C). As for the impacts of CREB in eNOS/AKT pathway, WB experiment revealed that the up-regulation of CREB reduced the phosphorylation of eNOS/AKT pathway in HUVECs induced by insulin (P<0.001; Figure 5).

Discussion
MGO can be obtained from food or produced in the body. In vivo, the main pathway of MGO production is glycolysis, and its content is abnormally increased due to the DM or hyperglycemia [16].
As MGO is hyper-responsiveness with short half-life, as well as the human body contains a variety of enzymes degrading MGO, so MGO can be self-cleared by the body under normal physiological conditions [17]. In abnormal conditions, the obstacle of MGO clearing pathway will lead to the gradual accumulation of MGO content, which can not only directly cause damage to various cells in the body, but also induce the mass-generation of advanced glycation end products [3]. In previous studies, miRNAs have been proved to be closely related to the content of MGO, such as miR-30b [18], miR-214 [19], miR -9a-3p [20], etc. In this study, the expression of miR-450a-5p showed a downregulated trend in HUVECs with high-glucose status, which was also decreased with the increase of MGO concentration. Therefore, it can be speculated that miR-450a-5p might participate in the glycolysis process and play a role in the pathogenesis of DM. . In short, insulin has the effect of activating eNOS/AKT pathway and promoting the release of NO, thus preventing endothelial cells from damage, which is the same as the results of this study. On the contrary, we also found that the addition of MGO inhibited insulin-mediated eNOS/AKT pathway activation, whereas up-regulated miR-450a-5p eliminated the resistance of MGO to insulin. It could be seen that miR-450a-5p had a reverse effect on insulin resistance mediated by MGO.
For in-depth exploration, we discovered that CREB had a binding site with miR-450a-5p through prediction websites and double-luciferase reporter analysis, which demonstrated that CREB was the target gene of miR-450a-5p. Furthermore, miR-450a-5p reversed the CREB-promoting effect of MGO.
In the past, the initial research on the function of CREB mainly focused on the nervous system, and it is known that CREB had a wide range of biological functions including learning and memory regulation [28]. In recent years, the research field of CREB has gradually expanded; some scholars pointed out that activated CREB can promote the proliferation and migration of endothelial cells [29]. In addition, activated CREB promoted insulin resistance and broke glucose balance in the body [30]. In this study, we learned that CREB obviously restrained the phosphorylation of eNOS and AKT, prompting that CREB had an activation effect on the eNOS/AKT pathway. In related studies, Niwano K et al.
[31] also reported that CREB competitively bound to the cAMP/ATF reactive element, so as to regulate eNOS gene expression in endothelial cells. These finding prompted that miR-450a-5p might relieve MGOinduced insulin resistance via targeting CREB.

Conclusions
In conclusion, miR-450a-5p decreased obviously in endothelial cells with high-glucose and MGO of certain concentrations (500, 1000 μmol/L). Through in vitro cell experiments, we knew that MGO could not only intensify the activity of endothelial cells, but also accelerate cell invasion and fat accumulation, which could be reversed by up-regulated miR-450a-5p instead of down-regulation.
Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin.
Nevertheless, up-regulated miR-450a-5p eliminated the resistance of MGO to insulin via targeting CREB, which might be a potential therapeutic target to improve MGO-induced insulin resistance and benefit patients with related diseases.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no conflicts of interest.