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Identifier

000352385

Title

Μελέτη των micrornas του ανθρώπινου κυτταρομεγαλοϊού σε σχέση με πιθανούς κυτταρικούς στόχους

Author

Γουλιδάκη, Νεκταρία Μ.

Thesis advisor

Σουρβίνος, Γεώργιος

Reviewer

Καλαντίδης, Κρίτων

Abstract

MicroRNAs (miRNAs) are small RNA molecules that exert post-transcriptional regulation and can alter protein and/ or mRNA levels of their targets. Human cytomegalovirus (HCMV) has been reported to code for 17 mature miRNAs, so far. Here we set out to experimentally verify computational predictions for hcmv-miR-UL22A and hcmv-miR-UL22A* in relation to the cellular targets RHOB and RGL2, respectively. Tandem repeats of the RHOB and RGL2 putative minimal miRNA binding sites were cloned in different luciferase reporter vectors. In the case of RHOB, a 30-57% decrease in luciferase activity was observed in HCMV infected fibroblasts, whereas in the case of RGL2 no significant decrease was detected. A 688 bp fragment of a predicted RHOB 3’ UTR containing the microRNA target site, was also cloned into the luciferase vector exhibiting a 56-69% decrease in luciferase activity in infected fibroblasts. No decrease in luciferase activity of the RHOB miRNA binding site containing vector was observed in not infected fibroblasts and in HeLa and HEK 293T cells which are not permissive to HCMV. The two microRNAs were cloned in their common precursor form and in addition, hcmv-miR-UL22A in its mature form and luciferase assays and northern blot experiments were performed to evaluate their expression. Moreover, the issue of the alternative 3’ UTR of RHOB assumed to exist based on the prediction that hcmv-miR-UL22A may target RHOB, was addressed. Using immunofluoresence staining, it was shown that HCMV infection results progressively in an asymmetrical increased perinuclear localization of RHOB in fibroblasts. Additional experiments are required to elucidate the possibility of RHOB regulation by hcmv-miR-UL22A and the role of RHOB in the course of HCMV infection.

Physical description

85 σ. : πιν. ; 30 εκ.

Language

Greek

Subject

Cytomegalovirus

MicroRNAs

Issue date

2007-12-14

Collection

School/Department--School of Medicine--Department of Medicine--Post-graduate theses

Type of Work--Post-graduate theses