الفهرس 
COLORECTAL CANCEROnly 14 pages are availabe for public view
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Abstract
colorectal cancer ranks the third most common diagnosed
cancer worldwide among men and women as well as
occupying the third rank for causing cancer related mortality
worldwide. However, most of the diagnosed cases and deaths
were attributed to modifiable risk factors, such as unhealthy
diet, smoking, alcohol consumption, obesity and physical
inactivity which are potentially preventable.
In Egypt CRC is ranked the seventh among Egyptian
males or females with an increase in the estimated number of
diagnosed cases due to the paucity of epidemiological studies
performed on the Egyptian population and the deficiency of
effective screening programs as well.
Currently, the most widely used diagnostic approaches
for CRC are the endoscopic procedures, such as colonoscopy
and sigmoidoscopy with high sensitivity and specificity for
identifying polyps and cancers. However, high cost,
invasiveness and time-consuming procedures have resulted in
poor compliance rates. Some inexpensive and non-invasive
methods, such as the fecal occult blood test (FOBT) based
screening, have also been developed, but with lower sensitivity
and specificity. Also, the currently used tumor markers for
diagnosis of CRC; CEA and CA19.9 were proven to have a low
diagnostic applicability due to their relatively low sensitivity
and limited specificity. This signifies the marked need for
C
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introduction of new biological markers for screening, diagnosis
and prognosis of CRC.
TLRs; the pattern recognition receptors have shown
aberrant expression in CRC cells and immune cells of the
tumor microenvironment leading to immune evasion, immune
suppression and further malignant progression. Furthermore,
miRNAs were found to function as ligands of these TLRs
leading to their activation and hence induction of the TLR
signaling pathway restoring their function and expression.
In light of the 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 CRC together with the implication of
the immune system in CRC through TLRs. We retrieved TLRs
expressed in CRC (TLR1, TLR7 and TLR8) followed by the
retrieval of miRNAs that are expressed in CRC and might act
as ligands to the retrieved TLRs (miRNA-122, miRNA-29b and
miRNA-15b) from public databases. Then, insilico molecular
docking of selected miRNAs into selected TLRs was done
through HDock web server to predict the direct binding
possibilities between TLRs and miRNA ligands and hence
verify the effects of miRNA ligand activation of TLRs on CRC
pathogenesis or their use as a potential therapy of CRC.
Afterwards, we investigated the chosen markers‘ expression
and diagnostic significance in CRC patients, benign colorectal
neoplasm patients and healthy volunteers by ELISA for TLRs
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protein and qPCR for miRNAs. Results were assessed for
association with clinical features and laboratory parameters of
CRC patients.
This study was done at the Medical Biochemistry
Department, Faculty of Medicine, Ain Shams University during
the period from August 2018 – September 2020 and included
50 CRC patients, 25 benign colorectal neoplasm patients and
25 healthy normal volunteers.
The aim of the current study was to evaluate the clinical
utility of serum levels of TLR1, TLR7 and TLR8 expression as
non-invasive biomarkers in diagnosis of CRC by ELISA as
well as miRNA-122, miRNA-29b and miRNA-15b by
quantitative Real Time -PCR and to correlate the expression of
these markers to the various clinico-pathological parameters of
the patients. This was done in an attempt to evaluate their role
in CRC assessment and their use as potential selected
diagnostic biomarkers. Also, to characterize the efficacy of
miRNA mimics of miRNA-122, miRNA-29b and miRNA-15b
as suspected ligands of TLR1, TLR7 and TLR8 in LS174T and
HT29 CRC cell lines.
The study included 100 subjects classified into three main
groups:
group 1: 50 colorectal cancer (CRC) cases with median
age 50years.
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group 2: 25 benign colorectal neoplasm cases with
median age 51 years.
group 3: 25 Healthy normal individuals with median age
50 years.
All studied subjects in this study were subjected to full
clinical and radiological examination preceded by complete
history taking and routine laboratory investigations including
tumor markers as serum CEA and CA19.9.
Evaluation of TLR1, TLR7 and TLR8 expression in
serum samples was performed by ELISA in all samples while
evaluation of miRNA-122, miRNA-29b and miRNA-15b
expression in serum samples was performed by Real Time PCR
in all samples. Then the results were statistically analyzed by
the SPSS software.
We found that TLR1, TLR7 and TLR8 could be used as
sensitive biomarkers for early diagnosis of CRC with recorded
sensitivity 86%, 84%and 88% respectively, while the recorded
specificity was 92%, 90%, 92% and high accuracy of 89%,
87% and 90%. This makes them better than the currently used
biomarkers CEA and CA19.9 that are less sensitive (58% and
54%) and less accurate (72% and 71%) in diagnosis of CRC.
We also found that miRNA-122, miRNA-29b and
miRNA-15b could be used as sensitive biomarkers for early
diagnosis of CRC with recorded sensitivity 82%, 88%and 86%
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respectively, while the recorded specificity was 94%, 90%,
88% and high accuracy of 88%, 89% and 87%. This makes
them better than the currently used biomarkers CEA and
CA19.9 that are less sensitive (58% and 54%) and less accurate
(72% and 71%) in diagnosis of CRC.
There was a highly significant positive correlation shown
among all the investigated serum biomarkers; TLR1, TLR7,
TLR8, miRNA-122, miRNA-29b and miRNA-15b (p<0.01).
However, the classical tumor markers CEA and CA19.9 have
shown a highly significant negative correlation with all the
investigated serum biomarkers; TLR1, TLR7, TLR8, miRNA122, miRNA-29b and miRNA-15b (p<0.01).
Also, there was a highly significant positive correlation
between serum miRNAs; miRNA-122, miRNA-29b, miRNA15b and tissue miRNAs; miRNA-122, miRNA-29b and
miRNA-15b (p<0.01) verifying the tissue source of serum
miRNAs.
Furthermore, the results of this study showed the effect
of introducing miRNA mimics into LS174T and HT29 CRC
cell lines on the cell count, viability, expression of the studied
miRNAs and functional expression of TLRs.
Upon transfection with miRNA mimics, count and
viability of CRC cells significantly decreased in comparison to
untransfected, mock and negative control groups.
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There was a highly significant difference between
miRNA mimic-122 transfected group and mock group and
between miRNA mimic-29b transfected group and mock group
and between miRNA mimic-15b transfected group and mock
group (p<0.01) as regards fold changes of expression (RQs) of
miRNA-122, miRNA-29b and miRNA-15b.The three miRNAs
expression level became upregulated after transfection of
miRNA mimics and remained downregulated in untransfected
group.
Finally, we measured the level of IL6 as it‘s a
downstream signal in TLR signaling cascade and that would
signify the activation of TLRs by the introduced miRNA
mimics. We found a significant increase in IL6 level in groups
transfected with miRNA mimics compared to untransfected,
mock and negative control groups. This would reflect that these
miRNAs might act as direct ligands that bind to TLRs and
activate TLR-signaling pathway resulting in the release of
inflammatory cytokines such as IL-6.