الفهرس 
TYPE 2 DIABETES MELLITUS (T2DM)Only 14 pages are availabe for public view
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Abstract
Type 2 DM is a chronic metabolic disease characterized by
insulin resistance and beta cell dysfunction. It is an
extremely heterogeneous disease in which the insulin resistance
and beta cell dysfunction resulted from environmental and
genetic factor.
Type 2 diabetes is the most prevalent form of diabetes
worldwide and account for 90% of cases globally. It represents
a major public health threat and considered as a principal cause
of morbidity and mortality affecting almost 6% of the world’s
population. Patients with type 2 diabetes usually have insulin
resistance and relative insulin deficiency and are often
associated with a strong genetic predisposition.
It’s one of the fast growing healthcare problems in
Egypt. According to international diabetes federation, Egypt is
the ninth leading country in the world for the number of
patients with type 2 DM. The prevalence of T2DM in Egypt
was tripled over the last two decades with a significant impact
on morbidity, mortality, and health care resources.
Insulin resistance is an enormous health care problem
and the central pathogenic factor for the development of type 2
DM; in addition, it is also strongly associated with several
metabolic risk factors as obesity, hypertension and
dyslipidemia.Several noncoding RNAs were reported linked to insulin
resistance. Therefore, Therapeutic targeting of deregulated
genetic network responsible for insulin resistance may open a
new hope for management of type 2 DM.
Competing endogenous RNAs (ceRNAs) are RNA
transcripts that make communication with each other by
decreasing targeting concentration of micro-RNA (miRNA)
with the derepression of other messenger RNAs (mRNAs)
having the common miRNA response elements (MREs). The
ceRNA theory has been applied to elucidate the pathogenesis of
numerous diseases such as cancer, muscular dystrophy
neurodegenerative disease and diabetes. However, little is
known about potential role of ceRNAs in metabolic disease,
especially diabetes mellitus.
Due to previous stated information, we used an
integrative approach based on bioinformatics analysis together
with clinical validation to provide great insights into the
molecular mechanisms of IR in the development of T2DM.
This study was done at Medical Biochemistry
Department, Faculty of Medicine, Ain Shams University during
the period from March 2016 – October 2018 and included 123
T2DM patients and 106 normal volunteers.
The aim of the present study was to retrieve potential
LncRNA-RP11-773H22.4 associated ceRNA network from atabases followed by validation of this network in patients’
and cell line samples by quantitative Real Time-PCR and to
evaluate the efficacy of CRISPR/Cas9 gene editing tool as a
potential therapeutic strategy to modulate the expression of
deregulated network in type 2 DM.
The study included 229 subjects classified into two groups:
group 1: 123 cases of type 2 diabetes mellitus patients,
T2DM diagnosis was proved according to the American
Diabetes Association practice guidelines. Their ages ranged
from 35 to 83 years, with a mean of 54.69± 8.855 years, the
median of ages was 55 years.
group 2: 106 healthy normal individuals. Their ages ranged
from 36 to 69 years, with a mean age 53.29± 7.04 years, the
median was 53 years.
All patients were subjected to complete medical history
taking, full clinical, radiological examination and routine
laboratory investigations including: Complete blood picture
(CBC), Fasting Blood Sugar (FBS), HbA1c and HOMA-IR.
The expression of the identified genetic network was
measured by quantitative real time PCR (qPCR) in the
peripheral blood mononuclear cells obtained from patients’
whole blood samples and in lymphocytes cell line. The data
were normalized using the endogenous GAP-dh mRNA and
RNU-6 miRNA as reference controls. Then the results were calculated and statistically analyzed by the SPSS software
version 20.
A significant difference was observed in the positivity
rates of LncRNA-RP11-773H22.4, miR-3163, miR-1 miRNAs
RET and m-TOR mRNAs, which were 73.2%, 85.4%, 70.7%,
80.5% and 99.2% respectively, in the diabetic group. However,
among the normal control, the positivity rates of LncRNARP11-
773H22.4, miR-3163, miR-1 miRNAs RET, and m-TOR
mRNAs were 14.2%, 17%, 4.7%, 20.8% and 17% respectively
(p<0.01).
Using ROC curve analysis, ≥ 2.123, ≤ 0.626, ≤ 0.995, ≥
2.99 and ≥1.6) were set as cut off values for LncRNA-RP11-
773H22.4, miR-3163, miR-1 miRNAs RET and m-TOR mRNAs
respectively. They showed sensitivity of (73.2%, 85.4%,
70.7%, 80.5% and 99.2% respectively), specificity of (85.8%,
83%, 95.3%, 79.2% and 83% respectively), and accuracy of
(79.04%, 84.28%, 82.1%, 79.91% and 91.7% respectively).
There was a highly significant positive correlation
between expressions of LncRNA-RP11-773H22.4, RET, m-TOR
mRNAs, HbA1c and HOMA-IR, and highly significant negative
correlation between them and miR-3163, miR-1 miRNAs
expression among the two studied groups (P<0.01).
Human PBMCs isolated from whole blood samples of
diabetic and healthy individuals. The PBMCs were cultured and lymphocytes grew for 3-5 days, then Cas 9 knockout of
LncRNA-RP11-773H22.4 at the lncRNA-miRNA interaction site
was done in the cultured cells. Cell pellets were collected from
the following 3 groups: diabetics before Cas9 editing, diabetics
after 72 hours post-transfection by Cas9 targeting LncRNARP11-
773H22.4 and healthy group, cells were assessed for both
cell count, cell viability and for molecular assays.
In conclusion, the results of our study Support our
previous hypothesis that LncRNA-RP11-773H22.4, miR-3163
miRNA and RET mRNA act as ceRNA network and might play
an important role in the pathogenesis of insulin resistance and
T2DM. And could be potential therapeutic target by gene
editing tools as CRISPR/Cas9 technique to modulate the
expression of the deregulated network in type 2 DM.