Mirna 375

1: Disease name : Type 1 Diabetes
2: miRNA ID : miR375
3: A brief description of the miRNA-disease
relationship: Type 1 diabetes is a progressive autoimmune disease that is largely
silent in its initial stages. Yet, sensitive methods for detection of -cell death and
prediction and prevention of diabetes are lacking. Micro-RNAs (miRNAs) have been
found at high concentrations in body fluids. Here in this study we sought to determine
whether an islet enriched miRNA, miR-375, is a suitable blood marker to detect -cell
death and predict diabetes in mice
4: miRNA expression pattern in the disease state:

Type 1 diabetes (T1D) is characterized by autoimmune destruction of the insulinproducing -cells, resulting in insufficient -cell mass and insulin secretion to control
plasma glucose (1). Clinical symptoms of diabetes are usually evident once more than
70% of the -cell population is destroyed (2). There is an unmet need for a cost-effective,
reliable method to assess -cell death in prediabetic patients, because early detection of
ongoing -cell death may offer a larger time frame for therapeutic interventions (3).
Currently, the earliest sign of -cell autoimmunity is the presence of circulating
antibodies against -cell antigens, which often can be detected months or years before the
onset of clinical symptoms (4). The most common autoantibodies in prediabetic patients
are directed against glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like
protein (IA-2), and insulin (IAA) (4). Positivity for 3 to 4 autoantibodies is associated
with a risk of developing clinical T1D in the range of 60%100% over the next 5 to 10
years. However, not everyone with autoantibodies progresses to T1D, and these
measurements cannot detect ongoing -cell death (1).
Micro-RNAs (miRNAs) are a family of endogenously produced single-stranded RNA
molecules of 22 nucleotides in length, which in the last decade have emerged as key
regulators of gene expression and cell function (5). miRNA-375 (miR-375) is the first
identified islet-specific miRNA (6) and the most abundant miRNA detected in the islets
(7) that was shown to be important for maintaining normal - and -cell mass in mice (8).
Recently, several miRNAs have been found in mouse and human plasma and serum at
surprisingly high concentrations unlike typically labile DNA/RNA. Extracellular
miRNAs appear to be by-products of dead or dying cells and remain in the extracellular
space due to the high stability of the Argonaute2-miRNA complex (9) or the presence of
lipoprotein complexes (10) or vesicles (11), which may confer stability. Regardless of the
mechanism, these miRNAs can be readily detected and quantified by PCR with
sequence-specific primers, making them potentially useful as biomarkers. Given their
stability and ability to be precisely quantified by PCR, we hypothesized that the most
abundant, islet-enriched miRNA, miR-375, may be a sensitive biomarker of -cell death
and predictor of diabetes.
5:Detection method for miRNA expression:
Animals
C57BL/6 male mice were obtained from the University of British Columbia Animal Care
Facility and female NOD/ShiLtJ (NOD) mice were obtained from The Jackson
Laboratory (Bar Harbor, Maine). Mice were fed a chow diet (2918; Research Diets, New
Brunswick, New Jersey) and were housed with a 12-hour light, 12-hour dark cycle with
ad libitum access to food and water. Body weight and blood glucose were measured after
a 4-hour morning fast. Blood was collected from the saphenous vein with EDTA-coated
capillary tubes. All procedures with animals were approved by the University of British
Columbia Animal Care Committee and carried out in accordance with the Canadian
Council on Animal Care guidelines. C57BL/6 mice were rendered diabetic with a single
ip injection of streptozotocin (STZ) at a dose of 180 mg/kg. A subset of diabetic mice
were anesthetized with inhalable isoflurane to receive an sc insulin pellet implant
(Limbit; Linshin Canada, Toronto, Ontario, Canada). Diabetes was defined as 2
consecutive blood glucose measurements >10mM.
Islet isolation and culture
Human islets were obtained from the Irving K. Barber Human Islet Isolation Laboratory
(Vancouver, British Columbia, Canada). Mouse islets were isolated from C57BL/6 male
mice (6 weeks old) by injection of collagenase (1000 U/ml in Hanks' balanced salt
solution). Pancreata were incubated at 37C for 11 minutes, and islets were hand-picked
to >95% purity. Islets were cultured in Ham's F-10 containing 0.5% BSA, 6.1mM
glucose, 100 U/ml penicillin, and 100 g/mL streptomycin. The next day, islets were
plated on a 96-well plate (40 islets per well) and treated for 24 hours with a cytokine mix
(IL-1, 10 ng/mL; TNF-, 50 ng/mL; interferon [IFN]-, 1000 U/mL) or 2mM STZ (in
0.1M sodium citrate, pH 4.5). When indicated, z-VAD (50M) or PJ34 (10M) were
added 1 h before. Cell viability was assessed using the live/dead assay from Life
Technologies (Burlington, Ontario, Canada).
RNA isolation and quantitative RT-PCR
The human RNA panel was purchased from Life Technologies. Mouse RNA was
extracted from tissues of 8-week-old C57BL/6 mice. Tissues were homogenized with an
Ultra-Turrax, and RNA was isolated using miRCURY RNA isolation kit (Exiqon, Seattle,
Washington). Isolated RNA was treated with deoxyribonuclease (Life Technologies).
Extracellular RNA was isolated from 25 l plasma or 80 l cell culture media
(centrifuged for 5 minutes at 3000 g) in the presence of an MS2 carrier RNA (Roche,
Laval, Quebec) using a miRNeasy kit (QIAGEN, Mississauga, Ontario, Canada). RNA
was reverse transcribed with a Universal cDNA synthesis kit (Exiqon). Quantitative RTPCR (qRT-PCR) was performed using SYBR Green master mix (Exiqon) with LNAbased miRNA primers (Exiqon). Relative values were calculated with the CT (delta
threshold cycle) method.
Statistical analysis
All data are represented as mean SEM, and significance was set at P .05. Mouse
experiments were analyzed using a nonparametric Mann-Whitney t test and cell culture
experiments using a parametric Student's t test.
Hypothesis of the paper:
By detecting miRNA 375 , we can early detect diabetes type 1 and better therapeutic
interventions can be made.
Important findings of the paper:

The above data suggest that circulating miR-375 can be used as a marker of -cell death
and potential predictor of diabetes.Early diagnosis of diabetes may offer a larger time
frame for therapeutic interventions.

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1: Disease name : Type 1 Diabetes

4: miRNA expression pattern in the disease state:

5:Detection method for miRNA expression:

Hypothesis of the paper:

Important findings of the paper:

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