Supervisors info:
Σίδερης Διαμάντης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, Εθνικό Καποδιστριακό Πανεπιστήμιο Αθηνών
Σκορίλας Ανδρέας, Καθηγητής, Τμήμα Βιολογίας, Εθνικό Καποδιστριακό
Πανεπιστήμιο Αθηνών
Κοντός Χρήστος, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, Εθνικό Καποδιστριακό
Πανεπιστήμιο Αθηνών
Summary:
Introduction: Chronic lymphocytic leukemia is a malignant disorder characterized by the uncontrolled proliferation and accumulation of B lymphocytes in peripheral blood, bone marrow, secondary lymphoid tissues and the spleen. It is the most common form of leukemia in adults in Western countries. CLL can be easily diagnosed via blood tests results, however, it is still necessary to find new markers for early diagnosis. In addition, one of the key problems in the treatment of CLL, is the about choosing the appropriate treatment, something that can be achieved with the help of prognostic biomarkers, that will provide information on the overall survival of patients. MicroRNAs are endogenous non-coding small RNA molecules that have several important regulatory roles and functions in cells. Understanding the function of miRNAs in oncogenesis can provide information about key questions that still exist in cancer research. Increasing evidence suggests that large numbers of microRNAs are involved in the pathogenesis of CLL. Purpose: The main objectives of this thesis were: the qualitative and quantitative determination of the expression levels of microRNA-451a, microRNA-486-5p and microRNA-582-5p in patients with Chronic Lymphocytic Leukemia, with Real-Time qPCR, the confirmation or not of their association with overexpression or underexpression, comparing with healthy individuals but also among patients, compared to the overall survival, and their different clinical and pathological characteristics and, ultimately, the potential clinical significance of these microRNAs as diagnostic or prognostic biomarkers. Methods: Peripheral blood mononuclear cells (PBMCs) were separated with Ficoll from 82 samples of patients with Chronic Lymphocytic Leukemia and 29 Healthy Individuals, and total RNA was isolated from the cells using TRI REAGENT, followed by spectrophotometric determination of the concentration of the isolated Total RNA. Primers were then designed for microRNAs 451a, 486-5p and 582-5p, and cDNA synthesis followed, with reverse transcription PCR with an initial polyadenylation step (PAP RT-PCR). Finally, the expression levels of these microRNAs in these samples were quantitatively and qualitatively determined by Real-Time q-PCR, followed by Statistical Analysis, which examined the correlation of expression levels of these microRNAs, between CLL patients and healthy individuals, as well as among the various clinicopathological characteristics of the patients, and the overall survival (OS) for these patients. Results & Conclusions: The results of the experimental procedures reffered, initially helped quantify the expression levels of these microRNAs in the samples of patients with CLL and healthy individuals. The correlation of expression levels in these microRNAs between CLL patients and healthy individuals showed a different expression in the three microRNAs compared to healthy individuals, with statistically significant results(p <0.05). The correlation of the three microRNAs with the clinicopathological characteristics of CLL patients showed that there is a statistically significant correlation between microRNA-5825p and the widely used prognosis factor CD38, and also between microRNA-582-5p and the Rai clinical staging system. Finally, the overall survival of CLL patients in comparison to the three microRNAs, setting an optimal cut-off threshold, showed that in all three microRNAs, expression levels of positive and negative patients(according to the cut-off threshold), did not appear to be significantly different, with no statistically significant results on the overall survival (p> 0.05).
Keywords:
microRNAs, Chronic Lymphocytic Leukemia, CLL, PBMCs
