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92 ΠΕΡΙΛΗΨΗ
Τίτλος: Ανάλυση και μελέτη της έκφρασης σε καρκινικά κύτταρα, μέσω μη κωδικών μορίων RNA (ncRNAs), νέων εναλλακτικών μεταγράφων του γονιδίου PRMT1 με χρήση μεθοδολογίας αλληλούχισης νέας γενιάς.
Μαυρογιάννης Αδαμάντιος
Η αλληλούχιση νέας γενιάς αναμένεται να βοηθήσει σημαντικά στην ανακάλυψη της πολυπλοκότητας του εναλλακτικού ματίσματος και στην κατανόηση της σύνδεσής του με την καρκινογένεση. Ο σκοπός της παρούσας εργασίας ήταν να χρησιμοποιηθεί η τεχνολογία NGS για την εύρεση νέων εναλλακτικών μεταγράφων του γονιδίου PRMT1 σε ένα σύνολο καρκινικών κυτταρικών σειρών ανθρώπου με διαφορετική προέλευση. Για τον σκοπό αυτόν, απομονώθηκε RNA από 56 καρκινικές κυτταρικές σειρές ανθρώπου και συντέθηκε μονόκλωνο cDNA, χρησιμοποιώντας oligo-dT-adaptor ως εκκινητή. Μετά τη δημιουργία του cDNA, έγινε 3’ RACE-nested PCR για τη μοριακή κλωνοποίηση όλων των PRMT1 μεταγραφων. Όλα τα PCR προϊόντα καθαρίστηκαν και έγινε κατασκευή βιβλιοθηκών και ποσοτικοποίησή τους.
Ακολούθησε αλληλούχιση με τεχνολογία NGS σε ημιαγωγό με το μηχάνημα Ion Torrent Personal Genome Machine™. Με βιοπληροφορική ανάλυση των δεδομένων αλληλούχισης βρέθηκαν 19 νέες θέσεις συρραφής μεταξύ γνωστών εξωνίων του γονιδίου, που υποδηλώνουν την ύπαρξη νέων, μικρής συχνότητας, εναλλακτικών μεταγράφων του PRMT1. Επιπροσθέτως, η ανάλυση οδήγησε στην ανακάλυψη ενός νέου εξωνίου μεταξύ των εξωνίων 11 και 12 του γονιδίου, μήκους 90 νουκλεοτιδίων.
Η ύπαρξη κάθε νέου μεταγράφου επιβεβαιώθηκε με πειράματα PCR και ηλεκτροφόρησης σε gel αγαρόζης. Ως αποτέλεσμα, ταυτοποιήθηκαν 86 νέα μετάγραφα του PRMT1, από τα οποία τα 45 προβλέπεται να κωδικοποιούν πρωτεΐνη και 41 ως μη νοηματικά mRNAs, που μάλλον υφίστανται nonsense-mediated mRNA decay (NMD). Τέλος, η ρύθμιση της έκφρασης, μέσω μη κωδικών μορίων RNA (ncRNAs), των γονιδίων της οικογένειας PRMT μελετήθηκε μέσα από ένα δίκτυο αλληλεπιδράσεων μεταξύ micro-RNAs και γονιδίων στόχων, που κατασκευάστηκε σύμφωνα με τα δεδομένα της βάσης DIANA-TarBase v7.0.