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العنوان
Study of miRNA-370 and miRNA-375 Expression in Egyptian
Pediatric Acute Myeloid Leukemia patients /
المؤلف
Ali, Mona Mostafa Mohamed.
هيئة الاعداد
باحث / منى مصطفى محمد على
مشرف / وائل محمد السيد
مناقش / اسامة محمد احمد
مناقش / عبير حامد عبد الحلي
تاريخ النشر
2022.
عدد الصفحات
138 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
Physiology
تاريخ الإجازة
1/1/2022
مكان الإجازة
جامعة عين شمس - كلية العلوم - قسم علم الحيوان
الفهرس
Only 14 pages are availabe for public view

from 138

from 138

Abstract

Acute myeloid leukemia (AML) is characterized by abnormal proliferation and lack of differentiation of immature myeloid cells. In 2017 with the revised 4th edition of the WHO classification, AML was classified incorporating distinct genetic and molecular alterations, morphology, clinical and immunophenotype features.
MicroRNAs are a family of biological molecules including single stranded non-protein coding RNAs " ~ "21 nucleotides length and have been found to play important roles in a variety of biological processes of the hematological cancers including; development, embryogenesis, proliferation, organogenesis, apoptosis, and differentiation by modulating the expression of oncogenes or tumor suppressor genes. MiRNAs play an important role in the pathogenesis and diagnosis of AML. Much information is available about the role of miRNAs expression in adult AML diagnosis, but not in pediatric AML.
MiR-370 is located within the imprinted DLK1-DIO3 on human chromosome 14q32; this region was considered a cancer-related genomic region, suggesting the implication of miR-370 in carcinogenesis. MiR-370 was reported to have an oncogenic and a tumor suppressor role for a number of human malignancies.
MiR-375 is found between CRYBA2 and CCDC108 genes on human chromosome 2q35. MiR-375 plays an important role either as tumor suppressor or oncogenic miRNA. It may have an inhibitory role during the tumor development. Also, miR-375 promotes migration and invasion in lung cancer cells, glioma, and AML.
Because of the need for a non-invasive biochemical marker to diagnose pediatric AML and due to the presence of fluctuating data about the expression levels of miR-370 and miR-375 in different cancer types, this study aimed to investigate the expression patterns and the diagnostic relevance of miR-370 and miR-375 in newly diagnosed Egyptian pediatric AML patients.
The present study was conducted at Children’s Cancer Hospital, Egypt (CCHE-57357). A written informed consent was obtained from the legal guardians of all
patients and controls, after the approval of the Institutional Review Board (CCHE-IRB), according to national law and regulations.
The current study included 87 individuals who were divided into 2 groups:
a) Study group: included 72 patients aged less than 18 years with the diagnosis of de novo AML presented to the CCHE during the period 2013–2017.
b) Control group: included 15 age-sex-matched was randomly selected from healthy bone marrow transplantation donors.
• The study group was classified by morphological, cytochemical, immunophen-otyping, cytogenetic, and molecular testing.
• Detection of microRNA expression in peripheral blood by RT-QPCR. A total of 50 µg (per prep) of RNA was transcribed into cDNA using high quality TaqMan MicroRNA Reverse Transcription Kit and quantification of miR-370 and miR-375 expression was performed by TaqMan MicroRNA Assay mix.
• Each sample was examined in duplicate and the miRNA relative expression was evaluated using the 2-ΔCt, normalized to RNU6B and represented by median (Interquartile Range; IQR).
The expression levels of miR-370 was significantly downregulated in patients compared to normal controls (patients = 0.003 (0.0007 to 0.154); controls = 0.27 (0.078 to 0.479)) p<0.001. There was a significant correlation between miR-370 level and the TLC of the patients (p≤0.05). There was no significant association between miR-370 with any other clinical parameters. The ROC curve showed AUC of 0.966 for miR-370. MiR-370 level at 4.191 was the clear cut-off value to screen pediatric AML patients from controls. Based on this cut-off value, the sensitivity and specificity of miR-370 for distinguishing AML was 96.9% and 93.3%. At specificity 100%, miR-370 at cut-off 7.398 showed sensitivity of 72.3%. Logistic regression analysis model was performed. It revealed that miR-370 is associated with significant increased risk for AML (OR=2.432, 95% CI 1.441–4.105, p≤0.001).
The expression levels of miR-375 was significantly downregulated 0.0011 [0.0003 to 0.009] in comparison to control 0.0053 (0.0016 to 0.009) p<0.001. There was no such correlation regarding the miR-375 with TLC (p=0.871). There was no significant association between miR-375 levels with any other clinical parameter. The ROC curve showed AUC of 0.858 for miR-375. MiR-375 level at 9.284 was the clear cut-off value to screen pediatric AML patients from controls. Based on this cut-off value, the sensitivity and specificity of the values for miR-375 were 75.4% and 80.00% repectively. At specificity 100%, miR-375 at cut-off 10.324 showed sensitivity of 50%. LR model was performed, and it showed that miR-375 did not reveal any significant association with AML risk (OR 1.140, 95% CI 0.578–2.247, p≤0.705).
Spearman’s correlation analysis showed a significant correlation between the expression levels of both miR-370 and miR-375 in pediatric AML patients (R=0.470, p≤0.001).
The previous data suggested that downregulation of miR-370 and miR-375 expression may have an important role in AML development, which drove us to predict their possible functionally enriched pathways in AML. However, miR-370 showed better potential and sensitivity to screen pediatric AML patients and more significant correlation with AML risk than miR-375.
The miR-370-3p, miR-370-5p and miR-375 miRwalk targets to the differentially upregulated genes in AML (adj p<0.05). In this study, 209 significantly upregulated AML genes were found, with 5 overlapped genes PRKX, ASPH, ZMAT3, SPATA6 and PLXNA4. This result suggests them as tumor suppressor miRNAs as indicated here by the bioinformatics analysis. The P53 and MAPK signalling pathways are enriched for the miRNA targets. Both pathways are known to be essential for pediatric AML development and progression, These results suggest that restoring the expression of miR-370 and miR-375 may improve the treatment outcomes in AML pediatric patients through regulation of these pathways.
Conclusion
Both of miR-370 and miR-375 are significantly downregulated in PB of pediatric AML. This expression profile may point to their potential roles as tumor suppressors in AML pediatric patients; a role that was supported by bioinformatics analysis. More interestingly, results demonstrated miR-370 has a better potential than miR-375 as diagnostic biomarker for pediatric AML screening. MiR-370 significantly correlated with AML risk and clinical parameters. Findings also suggest for the first time the positive correlation between both miR-370 and miR-375 expressions. Taken together, miR-370 is a promising non-invasive diagnostic and a possible prognostic biomarker for pediatric AML that merits further investigations.