Supervisors info:
κ. Διαμάντης Σίδερης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας , ΕΚΠΑ
Summary:
Chronic lymphocytic leukemia is a hematological malignancy characterized by the accumulation of B lymphocytes. It is a type of slow-growing leukemia and mostly affects older people. The pathogenesis of the disease involves various genetic and molecular changes, the presence of which varies in patients. Ribonucleases are essential enzymes involved in RNA metabolism and play important role in many cell functions. Various studies highlight the involvement of these enzymes in carcinogenesis and analyze their possible use as prognostic biomarkers or as therapeutic targets. Ribonuclease κ (kappa) is an endoribonuclease which is expressed in all tissues and at all developmental stages. Its biological role it’s still unknown, however it seems to be involved in virus’s entry into host-cells via endocytosis. Recently, new generation sequencing experiments have led to the discovery of eight novel RNase κ alternative transcripts.
The aim of this master thesis is the study of the expression levels and clinical evaluation of RNase κ transcripts in patients with chronic lymphocytic leukemia (CLL). The research focused on the main transcript and on the alternative transcripts 7, 9, 14 and 20. For this purpose cancer cells lines were cultured in order to be used as calibrators in real time qPCR experiments. A total of 84 RNA samples isolated from peripheral blood mononuclear cells of 64 CLL patients and of 20 healthy individuals. Then, the cDNA was synthesized by reverse transcription. Quantification of the expression levels of RNase κ transcripts was performed by real time qPCR using the fluorescent dye SYBR Green I. Statistical analysis was carried out using SPSS program in order to compare the transcripts’ expression levels between CLL patients and healthy individuals and also to correlate the expression levels with the patients’ clinicopathological features. In addition, Kaplan-Meier survival analysis was performed.
Statistical analysis showed that the expression levels of the main transcript and transcript 20 were significantly reduced in CLL patients compared to healthy individuals. In contrast,
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transcript 9 shows a significant increase in expression levels in patients compared to healthy. Expression levels of transcripts 7 and 14 showed no significant difference between leukemic and non-leukemic population. ROC curves revealed the use of the main transcript and transcripts 9 and 20 as potential biomarkers to distinguish CLL patients from healthy individuals. Correlation analysis between transcripts’ expression levels and clinicopathological features of patients with CLL, revealed that the main transcript displays a significant negative correlation with the high white blood cells and lymphocytes count. Kaplan-Meier survival analysis showed that none of the five RNase κ transcripts has any potential prognostic value. That’s because there was no significant difference in overall survival between patients with low and patients with high expression levels of a particular transcript.
In conclusion, only the main transcript and alternative transcripts 9 and 20 can be correlated with chronic lymphocytic leukemia. Also, none of the transcripts could be used as prognostic marker in overall survival of CLL patients. However, transcripts need to be studied in larger CLL sample size in order to improve the reliability of the results.
