Use of miR-34a, miR-126, and miR-125a-5p expression level as diagnostic factor and therapeutic target for α-L-guluronic acid and β-D-mannuronic acid in Type-I diabetic rat models

Type 2 diabetes mellitus (T2DM) affects microRNAs (miRNAs) expression that are known as Post-transcriptional repressors of gene expression in plants and animals. This research aimed to investigate the expression levels of miR-34a, miR-126, miR-125a-5p, and miR-29b for diabetes diagnosis, and the effect of α-L-guluronic acid and β-D-mannuronic acid supplementation on the expression pattern of these miRNAs in diabetic rats. Total miRNAs were extracted and evaluated by qRT-PCR method. body weight, fasting insulin levels, food intake and Vitamin D level in four group after 4-week intervention also measured. The analyzes showed significant increase in expression levels of miR-126 and miR-125a-5p, in diabetic control groups (p=0.001). Also, miR-29b was undetectable in the sera of experimental rats. After treatment with β-D-mannuronic acid, the expression level of miR-34a was upregulated in diabetic rats (p=0.026), but α-L-guluronic acid was not significantly effective. Also, expression levels of miR-126 (p=0.026) and miR-125a-5p (p=0.041) were downregulated significantly in diabetic rats, after treatment with β-D-mannuronic acid and, α-L-guluronic acid respectively. these results suggest that use of miR 126 ,125a-5p and miR 34a for diagnosing diabetes is sensible, also we can consider β-D-mannuronic acid as new therapeutic agent for correct disturbance in miRNA profile of diabetic rats.


Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, fat, and protein metabolism due to a defect in insulin action and secretion (1). Major risk factor for cardiovascular disease exists in subjects with a history of Type 2 diabetes and most patients with diabetes die from complications of atherosclerosis (2). Oxidized low density lipoprotein (ox-LDL) plays a major role in the start and progression of atherosclerosis, in a diabetes patient serum ox-LDL level increase with the length of diabetes, even though the patients LDL-cholesterol level is maintained in the normal range, ox-LDL produced from LDL in an oxidative stress situation (3). The previous study approved that ox-LDL accelerates atherosclerotic plaque formation and may be contributing to the exacerbate of microvascular complications in diabetes (4). Mir-125a-5p was found to interfere lipid uptake and to decrease the secretion of some inflammatory cytokines (interleukin-6, interleukin-2, tumor necrosis factor-α, transforming growth factor-beta) in ox-LDL-stimulated monocyte (5). in diabetic subject mir125a-5p upregulate by ox-LDL and inflammatory cytokine as a protect factor and interfere with their action(5, 6).
As mentioned above, one of the causes of death of a diabetic patient is atherosclerosis, which is exacerbated by ox-LDL. Alginate is commonly extracted from brown algae. Its chain is made of homopolymeric regions of β-D-mannuronic acid and α-L-guluronic acid.
The previous study approved that Na-alginate (at 600 mg·kg − 1 ) increased weight of diabetic rat significantly comparing to normal control, and decreases pre-prandial and postprandial glucose (7). Another study indicated that α-L-guluronic acid had immunomodulatory effect by decrease the expression of Toll-like receptor 2 (TLR2) and Nuclear Factor-kB (NF-kB)genes in Common variable immunodeficiency (CVID) patients (8).
The β-D-mannuronic acid as a novel herbal extract drug plays an important role in the management of inflammatory diseases (9). β-D-mannuronic acid molecule is an agent with the lowest molecular weight and has no toxicity compared with other anti-inflammatory drug (10).
MiRNAs are short endogenous RNAs (about 19-24 nucleotides) that repress posttranscriptionally gene expression in eukaryotes. There is evidence suggesting that miRNAs are involved in many of pathological processes (11). A recent paper has approved that the 4 expression of microRNAs profiles change in diabetes (12). The previous studies demonstrate that miRNAs are present in the serum and plasma of rats and other animals such as mice, humans, The levels of miRNAs in serum are reproducible, and stable among individuals of the same species (13). Up-regulation of miR-29b in cell line induced by ox-LDL involved in reducing the methylation of DNA and the migration of the primary human aortic smooth muscle cell (HASMC) which mediated epigenetic regulation, maybe considers as a novel mechanism in atherosclerosis (14). MiR-34a could induce apoptosis by inducing G0/G1 arrest in mouse pancreatic beta cells.
In addition, higher expression of miR-34a may be related to the pathogenesis of T2DM.
Then the serum level of miR-34a is a potential biomarker for T2DM diagnosis. Moreover, the expression of mi-RNA34a gene increases in human atherosclerosis plaque (15,16).
Macrophages, absorbed into the adipose tissue through C-C motif chemokine receptor-2 (CCR2), were postulated play a role in the development of insulin resistance in type 2 diabetes mellitus (17). In another previous study, upregulation of miR-125a-5p was observed in regulatory T (Treg) cells extracted from pancreatic draining lymph nodes (PLN) of T1D patients. TNFR2 (Tumor necrosis factor receptor 2) and C-C Chemokine Receptor type-2 (CCR2) were identified as miR-125a-5p target genes. For instance, Increase expression of miR-125a-5p in Treg cells of diabetes patients which result in the reduced expression of CCR2, thus limiting their migration and function in the pancreas (18). Also, previous studies showed that miR-126 expression level decreased in T2DM manifesting. It suggests reduced miR-126 is a predictor for the onset of T2DM.
Furthermore, the miR-126 level acts as an essential factor in the efferocytosis process of apoptotic myocytes by macrophages, in high glucose conditions (HG) such as diabetes (19,20).
Due to considering the high prevalence of diabetes and its complication in Iran and the world, as well as the lack of adequate studies on the effects of α-L-guluronic acid and β-Dmannuronic acid on serum levels of miRNAs, the present study conducted in the animal model of type I diabetes on Sprague-Dawley rats to determine the effectiveness of α-Lguluronic acid and β-D-mannuronic acid on four miRNAs (miR-34a, miR-126, miR-125a-5p, and miR-29b) expression level.

Experimental procedure
The rats were divided into four groups, 6 animals in each group as follow.

Group I
Healthy control rats was administered normal saline 0.5 mL Other injections were administered every-other-days.

Blood samples:
At the end of the study, after weighing and measuring blood glucose, rats were anesthetized with Ketamine and Xylazine and then sacrificed with chloroform. A peripheral blood specimen was drawn from each rat (6 mL/rat). EDTA was used as an anticoagulant.

miRNA extraction
MiRNAs were extracted from 400ml of plasma using Hybrid-R miRNA isolation Kit (GeneAll, South Korea) according to manufacturer's protocol. Concentration and purity of extracted miRNA were assessed by Thermo Scientific™ NanoDrop™ 2000/2000c Spectrophotometer.

Primers
Revers Transcription (RT) primer: First strand of cDNA was synthesized from miRNA templates using a stem loop cDNA synthesis primer, as described by Kramer (21). of the treatment group -(Ct miRNAs -Ct miR-16) mean of the control group.

Insulin, and Glucose assay
The glucose level was measured by glucose oxidase protocol and using glucose kit of the (biochemistry, Tehran, cat number: 117500). Insulin levels were measured using (DiaMetra, Perugia Italy, cat number: DKO076) ELISA kit.

Statistical analysis
Data were analyzed using Kolmogorov-Smirnov test. Due to non-normal distribution of data on gene expression and final food intake, non-parametric methods were used to analyze these variables. Kruskal-Wallis test and one-way ANOVA were used for between group analysis of these variables.

Result
Body and biochemical characteristics at baseline and after the interventions in healthy diabetic rats showed in Table 2. Body weight of Diabetic group treat with α-L-guluronic acid increased significantly compare to diabetic control (DC), but blood glucose did not decrease in DC rats after treatment. The results of one-way ANOVA test showed no significant difference in the initial weight (P = 0.879) and initial Food intake (p = 0.633) between the studied groups, but there was a significant difference in the final weight (P = 0/031) and the final food intake (P = 0/005). Table 2 Body and biochemical characteristics in Healthy and Diabetics control groups at baseline and after the interventions with α-L-guluronic acid and ß-d-mannuronic acid. To investigated the differences of miRNAs between diabetic and non-diabetic groups, four miRNAs related to diabetes were measured in the sera of experimental rat using qRT-PCR after four-week intervention. Kruskal Wallis test was used for analysis. This analysis showed differences in the expression levels of miR-126, miR-125a-5p and miR-34a, in sera between groups (healthy, and diabetic control groups, and two diabetic groups treated with α-L-guluronic acid and β-D-mannuronic acid. MiR-29b was undetectable in our rats. MiR-34a expression level was lower significantly in the diabetic group compared to the healthy group (p = 0.001). Furthermore, after treatment with α-L-guluronic acid and ß-Dmannuronic acid supplementation in diabetic rats, the expression level of miR-34a increased. Increase the level of miR-34a after treatment was significantly in ß-Dmannuronic acid group (p = 0.026), but was not significantly in treated group with α-Lguluronic acid (p = 0.177) (shown in Fig. 1).
MiR-126 significantly increased in the diabetic group compared to healthy control (p = 0.001). After treatment with α-L-guluronic acid, and ß-D-mannuronic acid supplementation mir126 expression downregulated. This change was significant in ß-D-mannuronic acid treatment group (p = 0.026), and was not significant in the α-L-guluronic acid treatment group (p = 0.177) (Show in Fig. 2). Also, the miR-125a-5p increase in the diabetic group compared to the healthy control group (p = 0.001). This change was decreased after treatment with α-L-guluronic acid, D-mannuronic supplementation. Fold change caused by α-L-guluronic acid was significant (p = 0.041), but caused by ß-D-mannuronic acid treatment was not significant (p = 0.124) (shown in Fig. 3).

Discussion
It was seen that epigenetic and genetic changes could involve in diabetes and progression of insulin resistant (23). Several studies revealed the involvement of miRNAs in key cellular activities including proliferation, differentiation, and metabolic integration and some disease like diabetes (24). Some websites like miRBase present about hundreds of human miRNAs. For the Selection suitable miRNAs for our study, we reviewed the relevant paper and selected the four miRNAs, which have been proven engage in T2DM. The major finding of our study is that miR-34a downregulate, and miR-125a-5p, and miR-126 increased significantly in the diabetic rats compare to the healthy rats. Furthermore, about these three miRNAs, after treatment with one supplement (ß-D-mannuronic acid) significantly improved diabetes in rats, but after treatment with another selective supplement (α-Lguluronic acid) improved diabetes in rats very little and not significantly. Also, we could not detect miR-29b in rats. This result confirm Zhang et al. (25), finding that miR-29b in the serum of rat is undetectable.
According to the report of Jahromi et al. (26), β-D-mannuronic acid is a novel immunosuppressive drug and able to modify toll-like receptors (TLR) signaling by suppressing the NF-κB and miR-146a as a new therapeutic approach. The previous study revealed that miR-34a may be related to the pathogenesis of T2DM, Liu et al. (15), suggested that miR-34a can induce apoptosis by inducing G0/G1 arrest in mouse pancreatic beta cells. Also, Kong et al. (27), have shown that in patients with a history of diabetes serum miR-34a was significantly upregulated compared to nondiabetics patients, but contrary to our prediction, in our study miR-34a expression decrease in DC Compared to HC(Healthy control). In this study, we showed that β-D-mannuronic acid could change miR-34a expression significantly but α-L-guluronic acid could not change miR-34a expression.
The previous study approved that Serum miR-126 was significantly lower in impaired glucose tolerance (IGT), impaired fasting glucose (IFG) subjects and T2DM patients than in healthy controls (20). Serum miR-126 was increased significantly in IGT-IFG and T2DM patients by diet therapy, insulin, and exercise for six months. miR-126 is highly enriched in endothelial cells and contributes to the repair and maintenance of angiogenesis, vascular integrity (28,29). Our results in contrast to the findings of previous studies showed that miR-126 in plasma of DC significantly upregulated compared to HC group, which probably reduction of mir126 involved in the pathogenesis and complication of diabetes (30). Also after treatment with β-D-mannuronic acid, the expression level of miR-126 decreased significantly compared to the diabetic control group that did not receive the treatment. This inconsistency may be due to differences in study time, Previous studies have included subject with a long history of diabetes and hyperglycemia, which may reduce the expression of mir126 and thus expose people to cardiovascular disease (29), while our study was designed over a one-month period mir126, which is considered a protective factor, may increase in the short term.
MiR-125a-5p regulated the expression level of genes in the mitogen-activated protein kinase (MAPK) signaling pathway that is important in T2DM pathogenesis. Ciccacci et al. (31), and Herrera et al. (32), showed that the level of this microRNA increased in human and rat with T2DM disease compare to the healthy individuals, respectively (31,32). Our results confirm the findings of these previous studies and reveal that miR-125a-5p significantly increases in DC compared to HC. Also, after treatment with α-L-guluronic acid, the expression level of miR-125a-5p decreased significantly compared to the diabetic control group that did not receive the treatment.
As observed in the previous study, alginate could decrease fasting plasma glucose in diabetic rats (7). Our study, in consistency with previous studies, has shown that supplementation with using α-L-guluronic acid and β-D-mannuronic acid can decrease fasting blood glucose but in controversy with previous study this effect it's not significant In this study, we confirm the previous finding that the body weight of diabetic rats increased after intervention with α-L-guluronic acid, ß-D-mannuronic acid supplementation reverse mir-34a downregulated by diabetes and downregulated mir126 which increased by diabetes and also α-L-guluronic acid decrease mir125a-5p that increased by diabetes.
Therefore, ß-D-mannuronic acid seems to be better therapeutic factor than α-L-guluronic acid to regulate the expression level of miRs and their target genes. Although these two factors increase fasting insulin significantly compare to DC, help to gain weight losted in diabetes and increase gene expression of mir125a-5p which may because of modulate inflammatory cytokine. ß-D-mannuronic acid can hope the more relevant cure in the future by co-treatment with other therapeutic agents. a miRNA regulates many mRNAs, and conversely, a mRNA is regulated by several miRNAs. it's reasonable to measure mRNA also and inflammatory cytokine but because budget restriction we couldn't do it and its biggest limitation for our study. Furthermore, provide a precise and early diagnosis of diabetes to delay progression and early intervention for minimizing complication is necessary.
Complex etiology, which involves, genetics, diet, and environmental factors, is as diagnostic and therapeutic challenges for DM. Currently, physician and health staff are 13 using traditional methods include levels of HbA1c and glucose; However, these biomarkers are not early diagnostic factors for DM (33). Hence, the discovery of the new therapeutic agent and biomarkers is necessary. In this article, we evaluated three microRNAs as the early diagnostic factors for DM.

Consent for publication
All authors approved the final version of this manuscript.