Supervisors info:
Χρήστος Κοντός, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Apoptosis is a form of programmed cell death in which cells that have accumulated mutations, have been infected with a virus or, in general, are considered perilous or unnecessary, are eliminated. It is a tightly regulated process, which does not leave behind traces of inflammation or stress and dysregulation of this procedure may lead to several diseases, such as cancer. The intrinsic or mitochondrial pathway of apoptosis is activated by intracellular stress stimuli and is regulated mostly by members of the BCL2 protein superfamily. Μembers of this superfamily can directly or indirectly enhance or suppress each other’s function and exhibit pro-apoptotic or anti-apoptotic roles. All members of this family share BCL2 homology domains (BH domains) that resemble the domains of the BCL2 protein, which was the first member of this protein family that has been identified in a type of B cell lymphoma. Based on this feature, the members of this family are divided into three categories: the anti-apoptotic proteins that contain four BH domains (BH1-BH4), the pro-apoptotic proteins effectors that contain three BH domains (BH1-BH3) and the pro-apoptotic proteins that contain only one BH3 domain (BH3-only). An intriguing and newly discovered member of the effectors’ group is the pro-apoptotic protein BOK, which in contrast to other effectors has all four BH domains. Few and controversial studies regarding its role in apoptosis have been conducted and its exact role in the intrinsic apoptotic pathway has not been fully elucidated yet. It is generally accepted that in case of endoplasmic reticulum stress or accumulation of misfolded proteins, BOK is no longer degraded in the proteasome and can induce mitochondrial outer membrane permeabilization and subsequent apoptosis either by itself or by promoting indirectly the oligomerization of the most crucial effectors BAX and BAK. Whether it can induce apoptosis by itself or not, as well as whether it regulates other normal cellular functions, such as the fusion of mitochondria, is not yet clear and needs further investigation. Circular RNAs (circRNAs) are created upon a unique back-splicing mechanism and can have several cellular functions. Their role ranges from binding microRNA molecules and RNA binding proteins to regulating transcription and being translated in order to produce novel protein isoforms. The aim of this dissertation has been the discovery of circRNAs deriving from the BOK gene in human epithelial cancer cell lines. The experimental procedure consisted of total RNA isolation from seven human cell lines, followed by reverse transcription of RNA to cDNA using random hexamers and a series of conventional PCR, Semi-nested PCR or Nested PCR experiments utilizing various divergent primers to discover potential circRNAs originating from the BOK gene. Ultimately, a novel circRNA has been identified using Sanger Sequencing and has been found to be greatly and specifically expressed in SK-BR-3 breast cancer cells. Subsequently, several computational tools have been utilized to predict its potential cellular function. Besides the experimental validation of its potential role, analysis of its expression profile in patient-derived breast cancer samples may reveal an original biomarker for this disease. Consequently, these findings may lead to the discovery of new therapeutics or biomarkers, and it is essential that the function and the expression profile of many more BOK-circRNAs are investigated in the future.
Keywords:
apoptosis, BCL2 family, BOK, circular RNA, circRNA, Sanger sequencing, back-splicing
