Dysregulated Circulating micro RNAs Markers: New Evidence into Expression Pattern in Children with T1D among Egyptian Population

Background: miRNAs are gaining access as promising markers for a variety of autoimmune disorders yet, deviations between individuals at risk or developed T1D remains to be thoroughly explored. Objective: to study the pattern of miRNA expression proling in plasma obtained from patients with T1D and the matched control subjects Patients and Methods: equally divided numbers of T1D patients (90) and apparently healthy-matched control children (90) were analyzed for their expression prole of plasma miRNAs; miR-101-5p, 146-5p, 21-5p, miR-375, miR-126, and Let7a-5p by reverse transcriptase (RT-PCR) through quantitative real time technique. Results: the two studied groups were signicantly different in respect to their biochemical parameters; FBG, 2hpp and HbA1c levels (p<0.05). Among the deregulated molecules, miR-101, miR-21 and-375 were highly expressed, whereas, miR-146-5p, miR-126 and miR-Let7a-5p showed signicant low levels of expression in patients compared to control subjects (p<0.05). MiR-101, miR-146 were signicantly correlated to age at diagnosis of T1D and disease duration respectively. Furthermore, miR-126, -Let7a-5p showed signicant negative correlation with meanA1C values, the matter that was conned by multivariate analysis. Conclusion: dysregulation of the analyzed six micro RNAs pointed out to their pivotal role to be important biomarkers for T1D development.


Background
Type 1 diabetes mellitus (T1DM) is essentially characterized by autoimmune destruction of pancreatic beta-cells by innate T lymphocytes & macrophages [1]. The disease is usually diagnosed at levels of betacells destruction exceeding 80-90% by the in ltrating immune system. T1DM development is slow, providing a potentially long window of time in which it is possible to clarify and theoretically treat candidate individuals at risk [2,3].
It's realized that about 80,000 children could develop the disease per year. Complications related to T1D vascular drawbacks have a big impact on quality of life, the morbidity and the mortality rates, posing enormous burden on the overall systems of health care. Diabetic nephropathy could also be a number one explanation for the existing end stage renal disease (ESRD) and surely augments the danger of cardiovascular diseases (CVD). In addition, it should be taken into account the seriously occurring diabetic retinopathy that warranted the possibility of blindness in adult times. Thus, it is urgently required to spot novel targets for quali ed treatment options and to get innovative noninvasive biomarkers reinforcing risk prediction, early diagnosis, and prognosis assessment [4].
These short (~22 nucleotides) non-coding microRNA molecules are shown be important participant agents that regulate the pattern of organic phenomenon underlying disease pathogenic mechanistic effects through a posttranscriptional way [5]. Generally, the miRNAs exerted functions passed through binding with the 3' un-translated regions (UTRs) of their speci c genes, leading to translational inhibition or direct degradation of the targeted mRNA with the resultant decrease in the protein expression [5,6]. The observed alteration in miRNA expression has been closely related to multiplicity of human in ammatory and autoimmune disorders [7,8]. Their estimated regulatory control of more than 60% of the protein-coding genes had consequently been linked to many diseases, including cancer, endocrine disorders and autoimmune diseases notably T1D [9].
MiRNA-speci c pro les were observed in PBMCs or serum from T1DM patients, and these important molecules seem to modulate mRNA expressions of the major T1DM autoantigens [10].On considering these aspects, the present study aimed to investigate the variable pattern of miRNA expression pro ling in plasma obtained from patients with T1D and the matched control subjects through quantitative realtime PCR.

Subject And Methods
This case-control study was prospectively conducted on 90 children with T1D, with mean age of (10.93 ± 4.51 years) having variable disease duration and variable degrees of glycemic control, who were diagnosed according to ADA criteria (Group I) [1].
A group of apparently healthy age and sex-matched 90children were served as controls (Group II) with mean age of (10.15 ± 2.56 years). All were enrolled from Pediatric Department in collaboration with Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Menou a University Hospitals, Egypt. Collection of demographic data, anthropometric measurements, treatment regimens and other clinical important parameters were done through viewing the medical sheet records.
Cases who were suspected of not diagnosed as T1D; MODY, T2D, or secondary diabetes mellitus, or with evidence of chronic systemic/rheumatic diseases, in ammatory disorders, recent febrile illness or on long-term steroid therapy were excluded from the study.
Upon approval of the study protocol from Ethical Committee of Menou a University in accordance to Helsinki II Declaration criteria, a written informed consent was obtained from all participants in the study.
Following complete history taking & thorough clinical examination, all studied subjects underwent sampling of 7-10 ml whole blood after 12h overnight fasting via sterile techniques and divided into tubes as; One ml of blood was transferred into sodium uoride tube and another sample of blood was obtained after 2 hours for enzymatic colorimetric determination of blood glucose, using Spinreact kit, SPAIN [11].
Another 4ml of blood were transferred into two EDTA tubes: one of them was used for quantitative colorimetric determination of glycated hemoglobin expressed as percentage of the total hemoglobin by the use of kits supplemented by Teco diagnostics, USA [12], where A1c values ≥ 6.5% were the limits for diagnosing T1DM [1].
For molecular analysis, 2ml of blood were transferred into the other EDTA-containing tube and centrifuged for ten minutes at (4000) r.p.m. The clear supernatant was separated and kept frozen at -80°C until further processing. Determination of MicroRNA levels were applied throughout a sequence of orders with the objective of obtaining c. DNA via reverse transcription of previously isolated RNA together with measurement of MicroRNA levels using speci c primer sets after being referenced to endogenous control U6B. These steps were applied as:

RNA isolation:
A total RNA including also miRNA molecules was purely extracted from plasma using Qiagen™ RNA Blood Mini Kit (Qiagen, USA, 2013) as de ned by the manufacturer's instructions. HiSpec Buffer, 2μl 10×miScript Nuclease Mix, 2 μl RNase-free water, 2 μl miScript Reverse Transcriptase Mix, then a 10 μl Template RNA to reach a total reaction volume of 20 μl. Reverse transcription was carried out at 37°C for 60 minutes and 95°C for 5 minutes on Applied Bio systems 2720 thermal cycler (Bioline, Singapore, USA). Diluted cDNA was the template for 2 nd step real-time PCR using Qiagenproduced SYBR Green miScript kit. Adding universal Primers was based on mRNA sequences delivered from the miR-database for (mi RNA 101-5p, mi RNA 146a-5p, mi RNA-375,mi RNA 21-5p, miR 126, and miR Let 7 a-5p) as shown in Table1(a). Each reaction for real-time PCR was completed to a nal 25μL volume, as followed: 12.5 μl 2x QuantiTect SYBR Green PCR Master Mix, 2.5 μl 10x miScript speci c Primer, 2.5 μl 10x miScript primer assay, 4 μl Template c DNA and 3.5 μl RNase-free water, the mixture was actually incubated at these conditions:95°C for 15 min (as initial denaturation), then denaturation at 94°C for 15 s duration, annealing for 30 s at a temperature of 55°Cand nal extension for 30 s adjusted at 70°C, for designed 60 cycles. Ampli cation of small RNA RNU6B was performed with each experimental sample as an endogenous control. Data analysis was done in the real-time cycler Applied Biosystems®7500 software version 2.0.1 thermal cycler (Applied Biosystems, Foster City, CA, USA).
Validation of the quanti ed miRNAs: The relative quanti cation (RQ) of genes expression was really performed by comparative ΔΔCt method, in which the amount of targeted mi RNAs 101-5p, mi RNA146a-5p, mi RNA-375, mi RNA21-5p, mi RNA 126, and mi RNA Let 7 a-5p were normalized to RNU6B as an endogenous reference among patients and controls. It's to be noted that these miRNAs were chosen on the basis of evidence from available database and literature that showed association of these molecules to pathways involved in T1D development in human [2,9,13]. Demographic and clinical data of the studied groups were shown in (Table1).  Comparative results regarding the levels of micro-RNAs studied in both groups revealed that miR-101-5p, miR-21-5p, miR 375 were highly expressed in patients with T1D, with a difference of statistical signi cance (P<0.05), whereas miR146-5p, miR 126, and miR Let 7a-5p showed down-regulation of their plasma levels (p value <0.05) in order ( Table 2). Of the remarkable ndings in this study was that our results indicated a signi cant negative correlation of miR 101-5p with the age of onset (r=-0.264, p=0.015) and with the duration of illness of T1D (r=-0.162, p=0.02) in respect. MiR-146 was correlated with T1D disease duration (r=0.239, p=0.023). On the other hand, miR 126, and miR-Let7a-5p were signi cantly negatively correlated to mean T1D patients ' glycated Hb A1c levels; p value<0.05 (Table 3). Results of multivariate logestic regression analysis for T1D risk were shown in (Table 4), where miR126-5p and miR-Let7a-markers showed highly signi cant ndings after adjustment of values for age, sex and mean Hb A1C levels in patients group as evidenced by Odds ratio, CI 95% of 0.016(0.0 -0.544), p= 0.021for -126-5p and 1.808(1.006 -3.249) and p value=0.048 for mi-Let7a-in order.

Discussion
Emerging role of miRNAs in modulating gene expression has greatly developed, and are being recently implicated in the presentation of different diseases [14][15][16]. Validity Reliability in the level of expression of these circulating molecules favored the extreme ability to be recognized as key biomarkers of disease etiogenesis and progression status [17,18]. In fact, this was evidenced by the relation of these molecules to 60% or more of the coding genes that thought to be in linkage to various endocrinal and autoimmune diseases [19][20][21].
Based on the underlying autoimmune background of T1D, our studied cases with recent disease onset; not more than6-12months duration; were chosen upon their positivity for insulin autoantibodies, the matter that researchers related certain miRNAs molecules to be validated as newly developed markers at early phases. Although for some, the progressive pattern in at-risk individuals couldn't be clearly addressed, still there's several miRNAs are tightly associated with both glucose homeostasis and levels of autoantibodies to be cornerstone in risk strati cation [22].
In the issue of T1D, it's still tried to clearly advocate that the miRNAs have cornerstone step in T1D pathogenesis not merely markers of active B cell dysfunctional outcome [23]. Butz et al, reported a pivotal effect of mi-RNAs on pancreatic cellular biology, especially for B-cell differentiation, production of insulin, apoptosis and mediation of in ammation [24].
In our study, among the analyzed mi RNAs, miR-146a-5p, Let-7a, and miR-126 were found to be down regulated, whereas miR-101, 21-5p, and miR375a-5p were consistently up regulated in patients when compared to our control subjects.
As for miR-375, its extreme abundance in pancreatic tissue rendered it a re ection of B-cell mass and alterations in its metabolic functions [25]. In respect to our results, it was found that, the level of miR-375 in the plasma of patients with T1D was signi cantly increased. When globally analyzed for its correlation to HbA1C, no difference was noticed as evidenced by coe cient r value of 0.173 and p value of 0.201.
The relation of that molecule to meanA1C values were favored by Marchand et al., 2016 who found dysregulated miR-375 level in the blood of newly diagnosed children with type 1diabetes when quanti ed to high levels in human islet tissue, the matter that conferred that to be a hallmark in the etiology of T1D. Furthermore, it may be a marker of early phases of the disease [26].
Of the deregulated molecules in our study that was found to be up regulated in the group of T1D patients versus those of control subjects as a difference of statistical signi cance (p value<0.001, Table2)  Analyzing data from past literature, revealed that has-miR-21seemed to be highly expressed in plasma of T1D patients in comparison to controls. Osipova et al., 2014 [28] found similar results. Ongoing research related 21-5p to cytokines of in ammation. In addition to the ndings of Backe and coworkers [29], it was suggested that miR-21 overexpression was believed to in uence the Bax group/ apoptotic signaling pathway, hence inducing pancreatic B-cell death. This process could be served as a new therapeutically tried target for T1DM [30,31].
Another up-regulated miRNA in our study was -101-5p, that targeted reduction of insulin secretion and Bcell mass as a favor of its involvement in cytokine release regulation and altered signaling of STAT3, HGF/C-Met and Ephrin receptors pathway mechanisms . Adjuvant to our ndings of the signi cant association of miR101-5p to insulin autoantibody-positive cases of recent onset T1D was the largely analyzing study by Santos A et al., who reported that the expression of miR-101 was about 3 fold higher in patients with multiple autoantibodies levels [32].
As found in our study, the negative correlation of miR-101 with the age of onset of T1D (r= -0.264, p= 0.015), related studies suggested a greater rate of B-cell turnover and pancreatic injury in young children with T1DM [33,34].
Another important micro-RNA molecule that showed signi cant down regulation beside being indicated in patients with recent-onset T1D was the miR146 a-5p. This was evident through a lowered expression level in cases; median (IQR) of 0.16(0.02 -0.43) compared to levels of 0.77(0.0 -0.88) in control subjects (p<0.001). A possible biological effect explored from its consistent relation to genes linked to apoptotic and innate immune regulatory pathway mechanisms [35].
Let7 a-5p was one of the studied markers expressed at lower levels in the studied group of patients and demonstrated a statistically signi cant difference in comparison to that of controls. Similar results were evident in the study done by Tian C et a., where Let -7a was down regulated in both human and mice tissue derivatives [36].The later was known to be involved in the regulation of glucose metabolism .In agreement with our results, it was found to be negatively correlated to A1C by the study done by Erener et al., 2017 [37].
Assessing the level of miR-126 expression revealed contradictory ndings. Osipova et al., conducted lower urinary levels in patients with T1D, with no signi cant difference in plasma samples of the studied cases and control groups respectively [28]. However, the observation of Wang et al., clari ed decreased plasma levels of miR-126 in those with chronic ESRD [38]. Despite the disagreeable ndings identi ed in our study about the signi cant lower plasma level of the miR-126 in T1D patients to that of Osipova et al., they came in the same line with the proposed mechanism that related decreased level of miR-126 to deranged response to vascular endothelial growth factor (VEGF) and endothelial dysfunction [28,39,40].
In addition, previous reports considered this marker as a controlling factor for various biological processes [41][42][43], through linkage of decreased circulatory miR-126 levels to micro-vascular change and possibility of later-on long standing T1DM complications [43].
The noticed signi cant negative correlation between miR-126, mi-R Let7a-markers and high percentages of the mean HbA1C values suggested the signi cant association of the altered levels of the circulating miRNAs to hyperglycemic state [37]. These above considerations were nearly similar to the hypothesis of Akerman et al., 2018 who assumed that expression pro ling of miRNAs may be of value regarding their feasibility to be a distinguishing complementary markers in risky individuals with abnormal OGTT results [22].

Conclusion
Deregulation of microRNAs in our study revealed down-regulation of miRNAs 146-5p, 126-5p and Let 7a-5p molecules and the up-regulation of miR101-5p, 21-5p and 375-were identi ed in our study for their signi cant relation toT1DM. Additional signi cant negative correlation of miR126-5p and Let7a-5p micro RNAs with mean glycated HbA1C values is indicative of their possible use as biomarkers of hyperglycemia-associated pathophysiologic changes in T1DM.
Given the stability, reliability of these markers, they were preferred for their superiority over other quanti cation techniques establishment through q-RT-PCR and warranted our choice in that study to be the Declarations