Priprava plazmidov z vključenimi tarčami hsa-miR-375-3p in njihovo potrjevanje z luciferaznim testom v celičnih linijah HeLa in HOS

Kovšca, Živa (avtor), Ostanek, Barbara (mentor), Vrščaj, Lucija Ana (komentor)

miRNA so majhne nekodirajoče molekule, ki s svojim delovanjem vplivajo na številne fiziološke procese. Med njimi je tudi kostna prenova, kjer uravnavajo ravnovesje med razgradnjo in gradnjo kosti. Namen naloge je bil poiskati in ovrednotiti tarčne gene hsa-miR-375-3p, ki je bila v predhodnih študijah na Katedri za klinično biokemijo Fakultete za farmacijo identificirana kot diferencialno izražena miRNA pri izpostavitvi mezenhimskih matičnih celic osteoanabolni zdravilni učinkovini teriparatidu. Z bioinformatično analizo smo določili tarčna gena FZD4 in BMPR2, ki sta vključena v signalne poti kostne prenove. Zasnovali smo mutirane in nemutirane konstrukte obeh genov in jih vstavili v plazmide pmirGLO. V celice celičnih linij HeLa in HOS smo kotransfecirali plazmide z vstavljenimi konstrukti in mimike hsa-miR-375-3p. Z luciferaznim testom smo preverili interakcijo med hsa-miR-375-3p in tarčnimi geni. Interakcijo med hsa-miR-375-3p in FZD4 smo potrdili v celični liniji HOS, ne pa tudi v celični liniji HeLa. V celični liniji HeLa se je hsa-miR-375-3p z večjo afiniteto vezala na mutirano zaporedje BMPR2 kot na nemutirano zaporedje BMPR2. To nakazuje, da lahko ustvarjena mutacija gena BMPR2 predstavlja genetski dejavnik tveganja za razvoj kostnih boleznih. Medtem v celični liniji HOS nismo potrdili interakcije med hsa-miR-375-3p in BMPR2. Na osnovi naših rezultatov smo ugotovili, da hsa-miR-375-3p igra potencialno vlogo v kostni prenovi, saj je tvorila interakcijo z genom FZD4, ki je vključen v signalno pot Wnt/β- katenin, ki vpliva na razvoj in delovanje osteoblastov ter gradnjo kosti.

• Jezik: Slovenski jezik
• Ključne besede: kostna prenova, hsa-miR-375-3p, FZD4, BMPR2, luciferazni test
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-173006
• Datum objave v RUL: 12.09.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Preparation of plasmids with hsa-miR-375-3p targets and their validation using the luciferase assay in HeLa and HOS cell lines

Izvleček

miRNAs are small non-coding molecules that influence numerous physiological processes through their activity. Among these is bone remodeling, where they regulate the balance between bone resorption and formation. The aim of this study was to identify and evaluate the target genes of hsa-miR-375-3p, which was previously identified at the Department of Clinical Biochemistry, Faculty of Pharmacy as a differentially expressed miRNA upon exposure of mesenchymal stem cells to the osteoanabolic drug teriparatide. Through bioinformatic analysis, we identified the target genes FZD4 and BMPR2, which are involved in signaling pathways related to bone remodeling. We designed mutated and wild type constructs of both genes and inserted them into pmirGLO plasmids. These plasmids were cotransfected together with hsa-miR-375-3p mimics into HeLa and HOS cell lines. We used a luciferase assay to validate the interaction between hsa-miR-375-3p and the target genes. We confirmed the interaction between hsa-miR-375-3p and FZD4 in the HOS cell line, while no such interaction was observed in the HeLa cell line. In the HeLa cell line, hsa- miR- 375- 3p bound with higher affinity to the mutated BMPR2 sequence than to the wild-type BMPR2 sequence. This suggests that the introduced BMPR2 gene mutation may represent a genetic risk factor for the development of bone diseases. In contrast in the HOS cell line, no interaction was observed between hsa-miR-375-3p and BMPR2. Based on our results, we concluded that hsa-miR-375-3p potentially plays a role in bone remodeling, as it interacted with the FZD4 gene, which is part of Wnt/β-catenin signaling pathway that primarily influence the development and function of osteoblasts and bone formation.

• Ključne besede: bone remodeling, hsa-miR-375-3p, FZD4, BMPR2, luciferase assay