Downregulation of miR-451a in plasma may promote prothrombin expression and contribute to high blood coagulation state in gestational diabetes mellitus

Purpose: To investigate F2 and miR-451a levels in gestational diabetes mellitus (GDM) patients, evaluate the potential role of miR-451a in GDM, and to explore its possible downstream targets. Methods: Thromboelastography (TEG) analysis was conducted to analyze coagulation differences between the GDM and control groups. Quantitative polymerase chain reaction (qPCR) and immunoblot were performed to analyze miR-451a and F2 levels after exposure to high glucose and miR-451a mimics or an miR-451a inhibitor. Luciferase assay was used to explore the potential regulatory mechanism. Results: Enhancement of the patients' blood coagulation ability and down-regulated miR-451a expression levels occurred in the plasma of GDM patients. The data further show that hepatocytes exposed to high glucose reduced miR-451a levels and enhanced F2 expression. The overexpression of miR-451a inhibited the expression of F2. Conclusion: MiR-451a may serve as a promising marker for hypercoagulable state GDM patients


INTRODUCTION
Gestational diabetes mellitus (GDM) is a common complication in pregnancy [1] that may lead to embryo defects, abortion, abnormal fetal development, and premature delivery [2]. Blood coagulation enhancement is common in women in the third trimester of pregnancy [3]. Thromboelastography (TEG) could reveal the specific characteristics of hypercoagulability of blood during pregnancy. Hypercoagulability of blood is harmful for human bodies which can induce venous thrombosis in pregnant and parturient women [1]. In severe cases, repeated abortion and premature delivery may occur, which may result in maternal deaths [4]. Pregnant women may experience blood coagulation enhancement. Similarly, some diabetics also have blood coagulation enhancement.
Prothrombin is a serum clotting factor that is synthesized in the liver and dependent on vitamin K [5]. Factor Xa complex acts on prothrombin, breaking the two bonds on prothrombin, forming thrombin F1 and F2 (F1+2) [5]. Thrombin F1+2 reflects the activity of factor Xa complex and is a specific molecular marker for the activation of prothrombin [6]. Recent studies have found that elevated plasma F1+2 levels are associated with the presence of atherosclerosis and the risk of clinically symptomatic cardiovascular disease [6]. The expression of prothrombin is closely related to hyperglycemia [7]. MicroRNA, with lengths of 18-22 bp [8] interacts with the 3'-non-transcriptional region of the genes [8]. Several studies have found that microRNA levels were changed in patients with GDM [9]. For example, upregulation of miR-503 in GDM led to impairment of endothelial cell function [10]. MiR-222 regulates the expression of ER, thereby regulating insulin resistance in GDM [11]. In addition, studies have also shown miRNAs have important regulatory effects on blood coagulation [9]. MiR-451a expression is down-regulated in the placental exosomes of patients with GDM [12]. Herein, the data showed that the expression level of miR-451a in the plasma of GDM patients was down-regulated while blood coagulation was enhanced. Therefore, circulation of miR-451a was speculated to be closely related to the blood coagulation ability of GDM patients.

EXPERIMENTAL Thromboelastography
Ethical approval was obtained from the Ethics Committee of the Third Affiliated Hospital of Soochow University. A total of 44 Gestational diabetes mellitus (GDM) patients and 32 normal pregnant women underwent thromboelastography treated at The Third Affiliated Hospital of Soochow University using TEG @ 5000 Thromboelastograph Hemostasis Analyzer System (Haemonetics, Braintree, MA, USA). Blood was retrieved with the two-syringe method. Subsequently blood was citrated with 0.2 M solution of calcium chloride and added to a warmed cuvette. Thromboelastography parameters, including time r (minutes), time k (minutes), α angle (degrees), and maximum amplitude (millimeters) were recorded.

Cell culture and transfection
The HepG2 cell line was purchased from the American Type Culture Collection and incubated in Dulbecco's modified essential medium supplemented with 10 % fetal bovine serum (Gibco, CA, USA). For glucose stimulation, HepG2 cells were cultured in complete medium supplemented with 50 mM D-glucose for 72 h. Synthetic miR-451a mimics, miRNA mimic negative control, miR-451a inhibitor, and negative control RNA inhibitor were purchased from GenePharma (Shanghai, China). Transfection was conducted using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols.

Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. For miR-451a detection, total RNA was transcribed to cDNA using the miRNA 1 st -strand cDNA synthesis kit (Vazyme, Nanjing, China) and further subjected to qPCR using a miRNA SYBR qPCR Master Mix kit (Vazyme). U6 snRNA was used as an internal control for miR-451a. The sequences of primers used in this study were Has-miR-451a forward:

Cloning and reporter assays
The 3' UTR of prothrombin mRNA was amplified with specific primers and inserted into XhoI and NotI sites of the pGL3 plasmid downstream of the Renilla open reading frame. The sequence of ACGGUU in the 3' UTR of F2 was mutated into GCCAAA. Mutagenesis in the 3' UTR of prothrombin was induced using the Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and was verified by sequencing.
and miR-451a mimic+pGL3-F2-MUT using Lipofectamine 2000 in complete medium, the cells were harvested and a dual-luciferase assay kit (Promega, Madison, WI, USA) was used to measure reporter Renilla and firefly luciferase activities in accordance with the manufacturer's protocols.

Statistical analysis
The data were expressed as mean ± SD and analyzed using GraphPad Prism (San Diego, CA, USA) (version 5.04). P < 0.05 was considered as statistically significant.

GDM patients exhibited enhanced blood coagulation and reduced miR-451a levels
To reveal the differences in coagulation function between GDM patients and control subjects, we performed thromboelastography (TEG) measurements. As shown in Figure 1A, the mean values of time R and time K were reduced in the GDM group, while the α angle and maximum amplitude were low in healthy, pregnant women (Figure 1 A). These results suggest that, as compared with the control group, GDM patients differed in the degree of fibrin formation, the state of dissolution, the firmness of coagulation, and the strength of elasticity. TAFI was reduced in GDM patients, and prothrombin fragment 1+2(F1+2) levels were elevated in the GDM group (Figure 1 B). qPCR analysis showed a relatively lower level of miR-451a in the GDM group ( Figure 1C). Thus, the enhanced coagulation function and reduced miR-451a levels were revealed in GDM patients. .

Hepatocytes exposed to high glucose increased the expression of F2
The reduction of miR-451a was previously observed in GDM patients. To further detect the effect of high glucose on miR-451a, an in vitro high glucose model was constructed. Lower miR-451a levels were observed in hepatocytes exposed to high glucose (Figure 2 A). In addition, the mRNA and protein levels of F2 were elevated in high glucose cell model (Figure 2 B and C). Therefore, high glucose levels led to an elevation of F2 and a reduction of miR-451a.

MiR-451a overexpression inhibited F2 expression
To explore the possible relationship between miR-451a and F2, we induced miR-451a overexpression in Hep G2 cells. qPCR was performed to verify overexpression efficiency. As shown in Figure 3 A, transfection of miR-451a mimics elevated miR-451a levels. The miR-451a overexpressed cells were subjected to normal or high glucose medium. miR-451a overexpression led to decreased F2 levels. High glucose treatment increased the F2 levels, which were abrogated by miR-451a overexpression (Figure 3  B). Moreover, the F2 protein levels yielded a similar pattern (Figure 3 C). Overall, the results suggest that miR-451a overexpression inhibits F2 expression.

MiR-451a directly targets F2
Potential targets of miR-451a, were screened for identification through TargetScan (http://www.targetscan.org/vert_72/). The 3'-UTR of F2 harbored a sequence that was complementary to miR-451a, as indicated by bioinformatics analysis (Figure 4 A). To confirm the previous data, a luciferase assay was conducted and the results revealed that F2 promoter activity was significantly decreased in miR-451a mimic transfected cells (Figure 4 B). However, mutant-type F2 yielded no significant difference in luciferase activity, indicating that F2 is the target of miR-451a. In contrast, miR-451a inhibitor promoted F2 levels (Figure 4 C). Moreover, F2 protein levels were enhanced in miR-451a inhibitor group (Figure 4 D). Taken together, the data suggest that miR-451a regulates F2 expression.

DISCUSSION
Hypercoagulability, also known as the prethrombotic state, is a complication of gestational diabetes, a maternal and infant health risk, and one of the primary causes of perinatal death [1]. Therefore, detection of changes in blood coagulation degrees in pregnant women with gestational diabetes, as well as the identification of key biomarkers and the development of effective therapeutic drugs, is of great importance in diagnosing and treating gestational diabetes related complications [4]. In this study, blood coagulation was found to be enhanced in patients with GDM, while expression of miR-451a was decreased. These results suggest that miR-451a might be an important biomarker of GDM and can potentially affect the progression of GDM. TEG reflects the whole blood clotting process of thrombus formation and degradation and, consequently, can compensate for deficiencies in traditional blood clotting laboratory examinations. In this study, TEG analysis was performed and coagulation differences between GDM and control groups were revealed. TEG can partially reveal the specific characteristics of changes in the coagulation status of pregnant women during pregnancy.
Compared with traditional coagulation laboratory tests, TEG is more sensitive and effective in the detection and management of obstetric bleeding.
In addition, high glucose treatment of hepatocytes was found to promote the expression of F2. The role of F2 in hypercoagulation is already known [12]. Other studies have confirmed the importance of F2 in patients with diabetes [14,15]. As was previously reported, prothrombin fragment F2 is a polypeptide fragment produced by the factor Xa complex after hydrolyzing prothrombin during blood coagulation [13]. This study suggested that the downregulation of miR-451a resulted in an increase of prothrombin expression. As found with miR-451a overexpression assays, the expression of F2 was decreased. Through quantitative PCR assays, immunoblot assays, and luciferase assays, miR-451a was shown to bind to the promoter regions of F2, thereby suppressing the expression of F2. Therefore, the downregulation of miR-451a may promote prothrombin expression.
Previous studies also indicated metabolic abnormalities in GDM patients. For example, in GDM patients, higher concentrations of HDL were observed in the third trimester of pregnancy, and higher triglyceride levels were found in both the second and third trimesters [14]. Therefore, the hypercoagulable status of GDM patients might be alleviated by targeting miR-451a. The function of miR-451a in a variety of physiological and pathological processes has been widely revealed [15]. For example, MiR-451a suppresses the growth and metastasis of papillary thyroid carcinoma cells by suppressing ZEB1 expression [16]. miR-451a is also involved in gastric carcinogenesis via the AKT pathway [17]. Another study showed that miR-451a suppresses the activation of MMP2 and MMP9 in cardiomyocytes upon pathological stress stimulation [18]. In this study, miR-451a suppressed prothrombin expression and contributed to a high blood coagulation state, suggesting a critical role for miR-451a in the physiological regulation of bodies.

CONCLUSION
An increased coagulation function and decreased expression of miR-451a occur in patients with GDM. In addition, high glucose treatment of hepatocytes promotes the expression of F2. Overexpression of miR-451a inhibits the expression of F2; therefore, miR-451a may be a promising marker for hypercoagulable state GDM patients.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Min Qiu designed the study, supervised the data collection, and analyzed the data. Hao Zhang interpreted the data and prepared the manuscript for publication. Zhimin Shangguan supervised the data collection, analyzed the data, and reviewed drafts of the manuscript. All authors read and approved the manuscript.

Open Access
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