Research of microRNAs expression levels serum of patients with epilepsy

Postgraduate Thesis uoadl:1506210 784 Read counter

Unit:
Κατεύθυνση Κλινική Βιοχημεία - Μοριακή Διαγνωστική
Library of the School of Science
Deposit date:
2017-05-04
Year:
2017
Author:
Papagrigoriou Maria-Eleni
Supervisors info:
Δ. ΣΙΔΕΡΗΣ, ΑΝΑΠΛ. ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΒΙΟΛΟΓΙΑΣ
A. ΣΚΟΡΙΛΑΣ, ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΒΙΟΛΟΓΙΑΣ
Δ. ΒΑΣΙΛΑΚΟΠΟΥΛΟΥ, ΑΝΑΠΛ. ΚΑΘΗΓΗΤΡΙΑ, ΤΜΗΜΑ ΒΙΟΛΟΓΙΑΣ
Original Title:
Διερεύνηση των επιπέδων έκφρασης microRNAs στον ορό ασθενών με επιληψία
Languages:
Greek
Translated title:
Research of microRNAs expression levels serum of patients with epilepsy
Summary:
RESEARCH OF MicroRNAs EXPRESSION LEVELS SERUM OF PATIENTS
WITH EPILEPSY
Papagrigoriou Maria Eleni, Biologist, University of Patras
ABSTRACT
Epilepsy is a disorder of the brain characterized by an enduring
predisposition to generate epileptic seizures and by the neurobiologic, cognitive,
psychological and social consequences of this condition as it was defined by the
International League Against Epilepsy (ILAE) in 2005. Recently though (ILAE
2014) epilepsy is also considered as a disease of the brain defined by any of the
following conditions: i) at least two unprovoked (or reflex) seizures occurring >24 h
apart, ii) one unprovoked (or reflex) seizure and a probability of further seizures
similar to the general recurrence risk (at least 60%) after two unprovoked seizures,
occurring over the next 10 years and iii) diagnosis of an epilepsy syndrome.
Epilepsy is one of the oldest conditions in humanity which affects individuals of all
ages. It has been reported that approximately 50,000,000 people worldwide have
epilepsy, with an incidence of 24-53/100,000 in developed regions, making it one
of the most common neurological diseases globally.
MicroRNAs (miRNAs) is a class of small non-coding RNA which regulates
gene expression by preventing the process of protein expression as they bind to
the 3’-UTR of the mRNA target. Α dysregulation in their expression has been
linked to a number of clinically important diseases including epilepsy. MiRNAs are
highly expressed in human brain relatively to other organs. Changes to brain
miRNA levels following prolonged seizures (status epilepticus) in animal models is
a fact. Pathogenic situations which have main role in epilepsy such as neuronal
death, inflammation and gliosis are under miRNAs’ control. They exist in a wide
range of tissues and extracellular body fluids including serum and blood plasma
and so they can be used for clinical purpsose as new biomarkers for
epileptogenesis or even for the prognosis of epilepsy.
In the present study the development and optimization of new molecular
assay methods for hsa-miR-34a and hsa-miR-146a levels in serum of patients with
107
epilepsy has been performed. For this purpose we used tumor cell lines DU145,
BT-20, AGS and DLD-1, as well as 24 serum samples from patients with drugresistant
epilepsy and 24 serum samples from patients with no drug-resistant
epilepsy. The experiment protocol involved the isolation of total RNA, the reverse
transcription of RNA to cDNA using miRNA-specific stem-loop RT primers and
quantitation of cDNA with quantitative real time PCR (qPCR), using TaqMan-MGB
probes. The cel-miR-39 was used as endogenous control.
The efficacy control of our method was conducted initially by perfoming
single miR-specific reverse transcription and then by multiple miR-specific reverse
transcription for cel-miR-39, hsa-miR-146a and hsa-miR-34a in all four cell lines of
our study. It was observed satisfactory amplification of our target-molecules either
the use of single- or multiple-miR-specific reverse transcription. However, negative
control samples revealed the enhancement of non-specific products, specially in
hsa-miR-34a requiring further optimization of the method.
Aiming the reduction of non-specific product in hsa-miR-34a assay method
we conducted optimization of the miR-specific primers concentration in reverse
transcription reactions. The amplification curves for the total of the concentration
that have been used were satisfactory. Yet negative control samples showed once
more the enhancement of non specific products for hsa-miR-34a, requiring further
optimization of the method.
Therefore more optimizations were performed initially for the concentration
of the universal R primer and then for the forward primer of the qPCR reaction,
aiming to the reduction of non specific product in hsa-miR-34a assay method. In
both cases the ability of the assay for hsa-miR-34a was confirmed in the total
range of the used concentrations, while a significant reduction for the non specific
product was observed.
The efficacy control of our method was evaluated also in serum samples
from patients with epilepsy and for the cell line DU145, using multiple miR-specific
RT for cel-miR-39, hsa-miR-146a και hsa-miR-34a. The efficacy of our method
was satisfactory all the concentrations tested. More precisely, hsa-miR-146a it was
observed satisfactory amplification in the vast majority of the serum samples
tested as well as for the cell line DU145.
108
After testing our method in 20 serum samples from patients with epilepsy, a
satisfactory efficicacy in samples tested following the same assay method was
confirmed. However, the significant amplification of non-specific products in similar
amplification levels with hsa-miR-34a in samples, indicated the lack of specificity in
our assay method and therefore we decided the design of novel miR-34a-specific
stem-loop RT primer and forward qPCR prime. The use of the new miR-34a
specific stem-loop RT primer did not resulted to the reduction of non-specific
product accumulation, while the use of the new forward qPCR hsa-miR-34a primer
did not produce suitable amplification curves.
In summary, we have developed and validated the methodology for the
quantification of hsa-miR-146a in serum samples from patients with epilepsy,
using cel-miR-39 as endogenous reference gene for normalization purposes. The
development of miR-34a specific assay was not satisfactory. Further studies using
larger number of patients are required, in order to improve validity for its
expression in serum samples from patients with epilepsy.
Main subject category:
Science
Keywords:
Research, microRNAs, expression levels, serum, patients, epilepsy
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
357
Number of pages:
139
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