Priprava plazmidov z vključenimi tarčami hsa-miR-20b-5p in njihovo potrjevanje z luciferaznim testom v sesalskih celicah

Prezelj, Anja (avtor), Ostanek, Barbara (mentor), Vrščaj, Lucija Ana (komentor)

Molekule mikro RNA postajajo vedno bolj zanimive za raziskovanje, saj se vpletajo v mnogo fizioloških in patoloških procesov v telesu ter tudi v procese kostne prenove. Porušena kostna prenova vodi v različne bolezni, med drugim tudi v osteoporozo. V predhodnih raziskavah na Katedri za klinično biokemijo so ugotovili spremenjeno izražanje hsa-miR-20b-5p v povezavi z zdravljenjem osteoporoze. Namen naloge je bil poiskati in eksperimentalno potrditi tarčne gene te mikro RNA. Na podlagi bioinformatične analize smo izbrali gena FZD3 in BMPR2. Zasnovali smo oligonukleotide, ki so vsebovali vezavno mesto za hsa-miR-20b-5p na osnovi zaporedij nukleotidov v informacijski RNA tarčnih genov. Poleg tarčnih zaporedij smo zasnovali tudi zaporedje za pozitivno kontrolo, ki je bila komplementarno zaporedje nukleotidov mikro RNA. Sledila je optimizacija procesa vnosa oligonukleotidov v plazmid pmirGLO. Celoten proces je obsegal restrikcijo plazmida, agarozno gelsko elektroforezo, ligacijo oligonukleotidov v razrezan plazmid in transformacijo celic Top10 s tem plazmidom. Pripravljene plazmide smo v procesu transfekcije vnesli v dve sesalski celični liniji (HOS in HeLa). Na teh celicah smo nato izvedli dvojni luciferazni test. Izmerili smo kresničkino in Renilla luciferazno aktivnost. Ti dve vrednosti smo med sabo delili, da smo podatke normalizirali, nato pa smo jih s pomočjo statističnega programa SPSS analizirali. Ugotovili smo, da se je hsa-miR-20b-5p v celični liniji HeLa vezala na zaporedji za FZD3 in BMPR2, s čimer smo potrdili njihovo direktno interakcijo. V celični liniji HOS ni bilo statistično pomembnih razlik med nobenim parom vzorcev, vzrok temu je bila najbrž slabša transfekcija, ki bi jo bilo v prihodnje treba optimizirati. Dokazana vezava hsa-miR-20b-5p na ta dva gena bi lahko bila v prihodnje uporabna osnova za razvoj novih terapevtskih možnosti zdravljenja bolezenskih stanj, kjer je cilj zmanjšanje tvorbe kostnine (npr. kostni rak), po drugi strani bi lahko to vezavo poizkušali zavirati, s čimer bi dobili nove terapevtske možnosti pri zdravljenju osteoporoze.

• Jezik: Slovenski jezik
• Ključne besede: BMPR2, FZD3, luciferazni test, hsa-miR-20b-5p, osteoporoza
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-169258
• Datum objave v RUL: 21.05.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Construction of plasmids with hsa-miR-20b-5p targets and their validation with luciferase assay in mammalian cells

Izvleček

MicroRNA molecules are becoming increasingly interesting for research, as they are involved in numerous physiological and pathological processes in the body, including bone remodelling. Disrupted bone remodelling leads to various diseases, including osteoporosis. Previous studies conducted at the Department of Clinical Biochemistry identified altered expression of hsa-miR-20b-5p in connection with osteoporosis treatment. The aim of this thesis was to identify and experimentally confirm the target genes of this microRNA. Based on bioinformatic analysis, we selected genes FZD3 and BMPR2. We designed oligonucleotides containing the binding site for hsa-miR-20b-5p, based on the nucleotide sequences in the mRNA of the target genes. In addition to the target sequences, we also designed a sequence for a positive control, which was a complementary sequence to the microRNA. This was followed by an optimization of the process for inserting the oligonucleotides into the pmirGLO plasmid. The entire process included plasmid restriction, agarose gel electrophoresis, ligation of the oligonucleotides into the digested plasmid, and transformation of Top10 cells with the recombinant plasmid. The prepared plasmids were then transfected into two mammalian cell lines (HOS and HeLa). A dual-luciferase reporter assay was performed on these cells. We measured firefly and Renilla luciferase activity. These values were normalized by calculating their ratio, and the data were subsequently analyzed using the statistical software SPSS. We found that hsa-miR-20b-5p bound to the sequences of FZD3 and BMPR2 in the HeLa cell line, confirming their direct interaction. In the HOS cell line, there were no statistically significant differences between any of the sample pairs, most likely due to inefficient transfection, which would need to be optimized in future experiments. The confirmed binding of hsa-miR-20b-5p to these two genes could serve as a basis for developing new therapeutic strategies for conditions where suppression of bone formation is desired (e.g., bone cancer). Conversely, inhibiting this interaction might provide novel therapeutic options for treating osteoporosis.

• Ključne besede: BMPR2, FZD3, luciferase assay, hsa-miR-20b-5p, osteoporosis