الفهرس 
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Abstract
V. Summary
CRC is a complex disease that develops as a consequence of both genetic and environmental risk factors. Several studies have established that miRNAs play critical roles in various biological processes and their dysregulation could result in multiple diseases, including CRC. Aberrant expression of miR-181a has been reported in several types of cancer and appears to be significantly associated with the clinical outcome of human cancer patients. Notably, genetic variants including SNPs or mutations in miRNAs encoding genes may alter miRNA expression and/or maturation.
Therefore, the current study was conducted to investigate the possible relation between the presence of SNPs in the MIR181A1 gene among CRC patients and the risk of disease development. Moreover, to elucidate how this presence may alter the expression level of miR-181a and its target proteins, together with the evaluation of hsa-miR-181a-5p expression level in sera of CRC patients and testing its potential clinical value.
This is a retrospective case-control study conducted on 192 blood and formalin-fixed paraffin-embedded (FFPE) tissue specimens selected from the pathologic files archive of subjects admitted to the Department of Medical Oncology, Faculty of Medicine, Tanta University, Egypt, in the period between 2015 and 2021 and underwent endoscopic biopsy. The selected specimens were classified according to the standard clinical, radiological, endoscopic, and histological diagnosis into 160 patients with CRC and 32 subjects with a normal colon. Stages and pathologic features of primary tumors were defined according to the American Joint Commission on Cancer criteria.
Written informed consent was obtained from each participant before the start of the study. This work was conducted per The Declaration of Helsinki and approved by the local Ethics Committee of Faculty of Medicine, Tanat University, Gharbia, Egypt.
All subjects were subjected to
• Full history and clinical examinations.
• Bioinformatics-based selection of hsa-miR-181a-5p
• SNP selection and genotyping of MIR181A1 rs12039395 using allelic discrimination (AD) assay.
• RNA secondary structure prediction.
• Hsa-miR-181a-5p expression analysis.
• Prediction of hsa-miR-181a-5p target genes in CRC.
• Determination of PTEN protein expression by immunohistochemistry.
The results of the study can be summarized as follows
• Compared to the control group, a higher number of CRC patients were hypertensive, smokers, and had CRC first-degree relatives.
• 80% of CRC patients complained from bleeding per rectum which may cause anemia as a consequence of the disease.
• As expected, CRC patients had a significant elevation in the serum levels of CEA and CA 19.9 compared to the control group.
• Most of the tumors in the patient group were found in transverse colon, sigmoid colon, rectum, and ascending colon; respectively. The majority of CRC patients suffered from stage II tumors with size ≤5 cm and moderately differentiated tumor cells.
• The bioinformatics analysis identified 621 miRNAs targeting the CRC pathway, among which hsa-miR-181a-5p was ranked the 7th most highly significant non-coding miRNA enriched in this particular pathway.
• Hsa-miR-181a-5p was identified to target CRC alongside multiple CRC-related pathways such as p53 and PI3K-Akt signaling pathways.
• The genotypic distribution of the polymorphism fitted HWE in both groups.
• Compared to controls, the G-allele was more prevalent with less frequency of the T-allele in the tissues of CRC patients. Accordingly, the GG genotype was predominant, whereas the GT and TT genotypes were less frequent in cancer tissues.
• The SNP was associated with decreased susceptibility to develop CRC under all the tested genetic models except for the recessive model.
• The in silico prediction of the hsa-miR-181a secondary structure revealed an altered loop formation, together with a difference in the minimum free energy between the primary miRNAs of the two alleles. The higher minimum free energy of the G-allele highlights a potentially less complex structure with greater accessibility for the transcriptional machinery.
• The base pair probability values of the G-allele were lower than those of the T-allele.
• Hsa-miR-181a-5p relative expression was significantly elevated in CRC tissues by almost four folds compared to the control group.
• Tissue samples harboring the G-allele, from either control subjects or CRC patients, exhibited a significant over-expression of hsa-miR-181a-5p compared to those carrying the T-allele.
• Compared to controls, the circulating has-miR-181a-5p expression was significantly over-expressed in CRC patients, where it was associated significantly with advanced-stage and poorly differentiated tumors.
• Over hsa-miR-181a-5p expression was associated with an increased risk of CRC after adjusting for age, sex, smoking habit, the presence of co-morbidity, and the presence of family history of the disease as potential confounders.
• Circulating hsa-miR-181a-5p showed a high diagnostic value for CRC. However, this ability was lower than the CEA and CA 19.9 diagnostic efficacy.
• A Venn diagram was generated to identify the overlapped target genes of hsa-miR-181a-5p between miRDB, TargetScanHuman Release 8, miRTarBase, and miRTargetLink 2.0 databases showing that 27 genes are shared between the four databases.
• Two pathways targeted by hsa-miR-181a-5p and involved in CRC (p53 signaling pathway and PI3K-Akt signaling pathway) in addition to pathways in cancer were displayed using a Venn diagram which revealed that 4 genes were overlapped between all three.
• At last, PTEN was found as the only commonality between the common targets of hsa-miR-181a-5p and the shared genes of the pathways involved in CRC.
• A computational investigation was performed to examine whether hsa-miR-181-5p can directly interact with PTEN mRNA. miRTarBase predicted the binding site of hsa-miR-181a-5p in the 3′UTR of PTEN mRNA. Moreover, RNA22 v2.0 predicted only one heteroduplex between hsa-miR-181a-5p and PTEN mRNA.
• IHC staining revealed that PTEN was mainly restricted to the cytoplasm of CRC tumor cells, unlike normal colonic mucosal cells that showed intense cytoplasmic and nuclear localization.
• PTEN expression level was significantly decreased in the CRC group compared to the control group.
• A significant over-expression of hsa-miR-181a-5p was observed in individuals having low PTEN expression in both control and CRC groups compared to their counterparts with high PTEN expression levels indicating an inverse relationship between hsa-miR-181a-5p and PTEN expression levels.
• The depletion of PTEN expression was significantly associated with older age, large tumor size, advanced stage, and poor differentiation.
• Interestingly, the depletion of PTEN expression was significantly associated with the presence of MIR181A1 rs12039395 G-allele.
In summary, the present study gives insight into the effect of miR-SNP in the MIR181A1 gene. The rs12039395 influences the risk of CRC development via modulating hsa-miR-181a-5p expression, which consequently targets PTEN and inhibits its expression. Furthermore, the results of the current work pointed out that up-regulation of hsa-miR-181a-5p is associated with higher susceptibility to CRC, and hsa-miR-181a-5p appears to be a good candidate as a biomarker in clinical practice to diagnose CRC patients. These findings may provide a new avenue for understanding CRC pathogenesis and pave the road for pharmacogenetic studies to establish personalized medicine based on individual genetic variations.