first described as an autoimmune disease in which autoreactive t cells destroy insulin - producing beta cells of the pancreas , it is now recognized that several potential environmental factors , including the possible role of chronic viral infections , can be causative agents . although it is important to diagnose diabetes as early as possible in order to prevent ketoacidosis , circulating autoantibodies against beta cell autoantigens are currently the only biomarkers available in the clinics . however , the presence of these autoantibodies does not always correlate with the loss of beta cell mass as positive individuals do not always develop t1d . in addition , recent access to pancreatic tissue from the npod biobank has allowed showing the existence of a significant amount of remaining beta cells in the pancreas of t1d patients several years after clinical diagnosis [ 4 , 5 ] . therefore , identification of new biomarkers in the early phases of the disease is necessary and represents an important challenge , as the disease is largely silent in its initial stages . in that context , blood mirna levels represent a new class of biomarkers for diagnosis and prognosis of several diseases and have emerged as new targets for treatments and interventions . mirnas are small noncoding rnas of 1922 nucleotides which act as negative regulators of gene expression , mainly at the posttranscriptional level . studies in the last 5 years have demonstrated that mirnas are not only found intracellularly but are also detected outside cells in various body fluids . remarkably , it has been found that some circulating blood mirna levels are proportional to the degree of severity of the pathology such as drug - induced liver injury , cardiovascular infection , cancer , alzheimer 's , inflammation , and metabolic diseases ( obesity and type 2 diabetes ) . in the context of t1d , 12 mirnas were found more concentrated in sera from children and adolescents with newly diagnosed t1d compared to sera from age - matched controls . among them , an association of mir-25 with improved glycemic control and better residual beta cell function was found , suggesting that this mirna could be used during early and intensive management of newly diagnosed diabetes to improve blood glucose control and reduce microvascular complications . another recent study involving pediatric t1d patients with duration of disease longer than 1 year revealed a significant deregulation of mir-21 , mir-126 , and mir-210 in plasma and urinary samples . as dysregulation of these mirnas has been shown in type 2 diabetes or renal diseases , their alterations in plasma of t1d patients may indicate ongoing endothelial dysfunction or preclinical kidney disease . taken together , these 2 studies provided a proof of concept that the concentration of circulating mirnas was affected in both newly or long - term diagnosed t1d already under insulin treatment . however , no data indicate whether these blood mirnas could be monitoring the course of the disease in the early steps . mir-375 is highly expressed in the endocrine pancreas ( islet beta cells and nonbeta cells ) and was first cloned from an insulin - secreting cell line min6b1 . in vitro studies have shown that mir-375 is involved in islet development , inhibits insulin gene expression in response to glucose , and regulates voltage - gated na(+ ) channels and the exocytotic machinery . ko - mir-375 mice are hyperglycemic and have elevated plasma glucagon levels . based on these data , we hypothesized that the level of mir-375 could be altered in the blood of t1d patients of recent onset . thus , in this study , we have validated this hypothesis by quantifying the level of mir-375 in the sera from children at onset of t1d ( before subcutaneous insulin treatment ) and in age - matched controls . we have also analyzed the global mirna profile in human islets exposed to high glucose concentrations to determine whether glucotoxicity might affect the level of mir-375 in beta cells . sera from 22 children at onset of t1d within two days after clinical diagnosis ( immediate insulin - requiring diabetes with at least one positive autoantibody ) and before subcutaneous insulin treatment and sera from 10 control subjects ( nonobese and nondiabetic child ) were collected in 2014 at the pediatric endocrinology department ( hospital femme - mre - enfant , bron , france ) . exclusion criteria for both groups were any febrile illness during the last month and chronic inflammatory disease . all patients and controls provided written informed consent for the collection of samples and subsequent analysis . clinical and biological characteristics of t1d children and controls are summarized in table 1 . whole blood was collected and allowed to clot for 30  min at room temperature . clot was removed by centrifuging at 2,000  g for 10 minutes in a refrigerated centrifuge . the resulting supernatant designated as serum was transferred into a clean polypropylene tube using a pasteur pipette . autoantibodies to glutamic acid decarboxylase ( gad ) , islet - antigen 2 ( ia2 ) , and zinc transporter 8 ( znt8 ) were quantified by elisa ( medizym , medipan gmbh , dahlewitz , germany ) and were considered to be positive for titers above 5 , 10 , and 15  ui / ml , respectively . hemolysis of sera ( 80  l ) was evaluated by spectrophotometry ( 450  nm ) ( biochemical department , lyon - sud hospital ) . human islets were obtained in collaboration with the geneva university hospitals cell isolation and transplantation center ( switzerland ) and from the unit mixte de thrapie cellulaire et tissulaire de grenoble ( france ) characteristics of the donors are summarized in table 2 . the use of human islet preparations for experimental research was approved by the institutional review board for clinical research of the departments of neurology , dermatology , anesthesiology and surgery of the university hospital of geneva ( cer number 05028 ) . islets ( purity at least 70% ) were processed as described previously   and cultured in a dmem medium containing 5.5  mm or 16.7  mm glucose and 5% ( vol./vol . ) fbs ( paa laboratories gmbh ) . 150  l of serum was transferred in an eppendorf tube and mixed with 450  l of trizol ls specially designed for liquid samples ( life technologies ) . then , 150  l of chloroform ( sigma aldrich ) was added and 20  fmol of synthetic cel - mir-39 ( ucaccggguguaaaucagcuug ) was spiked into the serum ( qiagen ) . tubes were centrifuged for 20 minutes at 12,000  rpm ( 4c ) . a fixed volume of aqueous phase ( 250  l ) was mixed with 250  l isopropanol and 2  l glycogen ( roche diagnostic ) and tubes were centrifugated for 30 minutes at 13,000  rpm . the pellet was washed with ethanol 75% and dried and the pellet resuspended in 16  l rnase - free water . a fixed volume of total rna ( 5  l for mir-375 and 1  l for mir-39 ) was used for reverse transcription ( rt ) reaction . rt reactions were realized with 0.15  l dntp , 1.5  l buffer ( 10x ) , 9  l rnase - free water , 0.2  l rnase inhibitor , 1  l multiscribe rtase , and 3  l of specific mir-375 rt primers ( life technologies ) . rt reactions were realized at 16c for 30 minutes , 42c for 30 minutes , and 85c for 5 minutes using a ptc-100 peltier thermal cycler ( mj research ) . at the end of the rt reaction . 5  l of rt reactions was mixed with 10  l of taqman universal pcr master mix ( 2 , ( life technologies ) ) , 4  l of rnase - free water , and 1  l of taqman mirna assay ( 20 , ( life technologies ) ) . pcr was performed on a rotor gene thermocycler at 95c for 10 minutes , followed by 40 cycles of 95c for 15 seconds and 60c for 60 seconds . all samples were run in duplicate ; ct ( cycle threshold ) values were automatically determined by the software . total rna was extracted from 3 independent human islet preparations by using tripure isolation reagent ( roche applied science , france ) . rna concentration was measured with a nanodrop spectrophotometer , nd-1000 ( thermo - fisher , waltham , ma , usa ) . profiling of mirnas was determined using the taqman low - density array cards a and b ( applied biosystems ) . briefly , total rna was reverse transcribed using taqman mirna reverse transcription kit ( applied biosystems ) in combination with the stem - loop megaplex primers in 7.5  l . next , 2.5  l of rt was mixed with 2.5  l of megaplex preamp primers and 12.5  l taqman preamp master mix in a 25  l pcr reaction and preamplified . the preamplified cdna was then diluted with 0.1x te ( ph 8.0 ) to 100  l . qrt - pcr was performed on a 7900ht thermocycler ( applied biosystems ) using the manufacturer 's recommended cycling conditions : 50c for 2  min and 95c for 10  min and then 40 cycles at 95c for 15  s and 60c for 1  min . cycle threshold ( ct ) values were calculated with the sds software using automatic baseline settings with assigned minimum ct threshold of 0.2 . ct values > 40 were excluded from data analysis . each card included 3 mirna endogenous controls and one mirna assay not related to human . for each card , quality controls were performed on the raw data by checking internal controls and using box plot diagrams . since the currently used normalization small rna mammu6 plotted in each card was not stably expressed in our different samples , we have used the mean expression level of all fully observed mirnas for normalization . group comparisons were made by using student 's t - test ( p < 0.05 ) on normalized data , to select the differentially expressed mirnas . this study aimed to evaluate whether the circulating level of mir-375 was altered at onset of t1d . therefore , we have quantified mir-375 in sera from children population at onset of t1d ( immediate insulin - requiring diabetes with at least one positive autoantibody ) before subcutaneous insulin treatment . t1d children had elevated hba1c ( 11.82%  2.15 ) and had at least one positive autoantibody for gad , ia-2 , and/or znt8 , confirming an ongoing autoimmunity to beta cells . as shown in figure 1 , the level of mir-375 was significantly lower in the sera of the 22 t1d children than in the sera of the 10 healthy controls ( mean normalized ct values : 30.61  0.20 versus 29.02  0.57 , p = 0.008 ) . we did not find any association between mir-375 serum concentration and hba1c , glycaemia , and the number of autoantibodies ( table 3 ) . then , we have analyzed the global mirna profile in human islets exposed to high glucose concentrations to determine whether glucotoxicity might affect the level of mir-375 in beta cells . as shown in supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/1869082 , 410 mirnas were detected in human islets and mir-375 was the most expressed one . nine mirnas ( mir-519a , mir-212 , mir-320b , mir-27a , mir-30d , mir-23a , mir-30d , mir-23a , and mir-10a ) and 5 mirnas ( mir-375 , mir-485 - 3p , mir-23b , mir-485 - 3p , mir-23b , mir-627 , and mir-1197 ) were up- or downregulated by glucose , respectively ( figure 2 ) . upregulated mirnas targeted important signaling pathways for islet homeostasis and regulation of beta cell mass . they were also involved in viral infections and cancer development ( see box 1 ) . downregulated mirnas were involved in pancreas secretion and in protein processing and degradation ( see box 1 ) . in addition , downregulated mirnas targeted genes involved in biotin metabolism and glycosphingolipid biosynthesis , two functions affected in t1d patients [ 22 , 23 ] . interestingly , mir-375 was downregulated in human islets exposed to high glucose concentrations , suggesting a close relationship between mir-375 and glucose ( figure 2 ) . more particularly , their use as biomarkers to monitor the fate and/or function of the pancreatic beta cells is attractive . in this study , we have found that mir-375 is strongly decreased in the serum of newly identified t1d children , before the initiation of subcutaneous insulin treatment . these data are in agreement with previous studies on animal models showing that plasma mir-375 is altered in streptozotocin - treated mice prior to the onset of hyperglycemia or 2 weeks before diabetes onset in nonobese diabetic mice . as mir-375 regulates insulin secretion and beta cell mass [ 16 , 17 ] , we postulated that its expression might be altered in beta cells concomitantly to its circulating level . in order to further interpret our data , we have determined , for the first time , the mirna profile of human islets incubated with high glucose concentrations . the abundance of mir-375 in the mirna profile of human islets found in this study confirmed its critical role in human pancreatic beta cells , mirroring its well - established role in rodent islet biology . we found that mir-375 was downregulated in human islets treated with high glucose concentrations . decreased functional beta cell mass is a hallmark of both type 1 diabetes and type 2 diabetes ( t2 dm ) and , interestingly , obese and pre - t2 dm patients have also decreased level of blood mir-375 compared to healthy controls [ 2628 ] . on the contrary , t1d and t2 dm patients with controlled glycemia have increased circulating levels of mir-375 [ 2931 ] . as we have previously found that beta cells can release mir-375 in extracellular environment , our data could suggest a possible link between circulating mir-375 levels and early alterations of islet integrity and functionality reflecting glucotoxicity in humans . however , this hypothesis has to be temporized as it is likely that only a small proportion ( 1% ) of circulating mir-375 is originated from beta cells , at least in mice . extracellular mir-375 pool is very heterogeneous and various cells and tissues can release this mirna . in human , mir-375 is also strongly expressed in gi - tract , adipose tissue , liver , adrenal , and brain . thus , altered circulating level of mir-375 at the beginning of t1d development could be also a general marker of metabolic alterations and/or inflammation associated with the disease , in addition to being representative of alterations of islet physiology . indeed , most individuals affected by t1d exhibit multiple features associated with impaired b- and t - cell functions [ 3537 ] . presently , islet transplantation offers a potential cure for t1d . beside beta cell exhaustion and/or apoptosis , recurrent autoimmune damage creates a serious challenge to successful long - term islet transplantation . the analysis of mirnas changes ( both in pancreas and blood ) may help to find early biomarkers for beta cell damage . we have previously shown that circulating whole - blood cells from newly diagnosed patients with t1d , as well as autoantibody - positive first degree relatives , had increased expression of ifn - induced genes . this fits well with the current knowledge of disease pathogenesis with the possible role played by viral infections . therefore , in addition to hyperglycemia and inflammation , expression of mir-375 might be also influenced by environmental factors such as viruses . in line with this hypothesis , mir-375 concentration in serum , our data demonstrated that mir-375 might be a potential biomarker at the onset of t1d that is also modulated in human islets exposed in vitro to glucose . it remains to determine whether this mirna reflects only metabolic disturbances observed during t1d or also reflects chronic processes leading to inflammation of the pancreas and beta cell loss . moreover , quantification of circulating mir-375 in prediabetic individuals is now necessary to validate the role of this new biomarker during the early phases of the disease .
background . 
 the use of mirnas as biomarkers for type 1 diabetes ( t1d ) risk is attractive as t1d is usually diagnosed in front of acute symptoms . as mir-375 is highly expressed in the endocrine pancreas , we postulated that its circulating level might reflect beta cell alterations and might be altered in the blood of t1d patients recently diagnosed . 
 methods . 
 sera were obtained from 22 t1d children at onset of the disease , before subcutaneous insulin treatment , and from 10 nondiabetic pediatric controls . mir-375 seric level was quantified by stem - loop rt - pcr - based assay . 
 mirnas regulations in isolated human islets in response to high glucose concentrations were determined by taqman low - density array . 
 results . 
 the abundance of mir-375 , among the 410 mirnas detected in human islets , mirrored its well - established role in rodent islet biology . 
 upregulated mirnas targeted genes involved in islet homeostasis and regulation of beta cell mass . downregulated mirnas , including mir-375 , were involved in pancreas secretion and protein turnover . 
 seric level of mir-375 was lower in t1d children versus age - matched controls , without any correlations with hba1c , glycaemia , and number of autoantibodies . 
 conclusion . altered circulating level of mir-375 at onset of t1d 
 might be a general biomarker of metabolic alterations and inflammation associated with the disease .