Ocena učinkov mikro RNA hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a in hsa-miR-31-3p na osteogeno diferenciacijo mezenhimskih matičnih stromalnih celic

Veljanovska, Melisa (avtor), Ostanek, Barbara (mentor), Vrščaj, Lucija Ana (komentor)

Na diferenciacijo mezenhimskih stromalnih matičnih celic (MSC) vplivajo številni epigenetski dejavniki, med katerimi imajo ključno vlogo mikroRNA (miRNA). MiRNA delujejo kot regulatorji izražanja genov, pomembnih za osteogenezo, in uravnavajo homeostazo skeletnega tkiva. Številne raziskave dokazujejo, da motnje v izražanju specifičnih miRNA niso povezane le z osteogenezo, temveč tudi z razvojem kostnih bolezni, kot je osteoporoza. Slednja predstavlja enega večjih javnozdravstvenih problemov, saj prizadene približno 200 milijonov ljudi po svetu. V magistrski nalogi smo preučevali vpliv izbranih miRNA (hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p in hsa-miR-31-3p) na osteogeno diferenciacijo MSC. MSC, ki smo jih uporabili kot celični model za raziskavo, so bile pripravljene z lentivirusno transdukcijo tako, da so se izbrane miRNA bodisi zvišano izražale bodisi so bile utišane. V teh celicah smo z metodo kvantitativne verižne reakcije s polimerazo v realnem času analizirali izražanje ključnih osteogenih označevalcev (ALPL, BGLAP, COL1A1 in RUNX2). Te rezultate smo kvantificirali z relativno kvanitifikacijo z metodo standardne krivulje in statistično ovrednotili s Studentovim t-testom ter jih primerjali z barvanjem po Von Kossa. Na podlagi rezultatov smo ocenili vpliv miRNA na osteogenezo MSC. Rezultati magistrske naloge potrjujejo hipotezo, da ima spremenjeno izražanje miRNA pomemben vpliv na osteogeno diferenciacijo. Med preučevanimi miRNA je hsa-miR-363-3p izkazala največji potencial kot biološki označevalec in/ali terapevtska tarča, saj smo z rezultati qPCR dobili statistično značilne razlike v izražanju osteogenih genov v primerjavi s kontrolnimi vzorci, prav tako pa so se rezultati qPCR skladali z rezultati barvanja po Von Kossa. Naši rezultati prispevajo k boljšemu razumevanju vloge miRNA v osteogenezi in njihovemu potencialu v diagnostiki ter terapiji osteoporoze. Kljub obetavnim rezultatom uporaba miRNA v klinični praksi ostaja izziv zaradi njihove nespecifičnosti, nestabilnosti in pomanjkanja učinkovitih dostavnih sistemov. Potrebne so nadaljnje raziskave na kliničnih vzorcih in modelih in vivo za boljše razumevanje ter klinično implementacijo miRNA.

• Jezik: Slovenski jezik
• Ključne besede: mikro RNA, kvantitativna verižna reakcija s polimerazo, osteogeneza, teriparatid, osteoporoza
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-169869
• Datum objave v RUL: 13.06.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of the effects of microRNAs hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a and hsa-miR-31-3p on the osteogenic differentiation of mesenchymal stem stromal cells

Izvleček

The differentiation of mesenchymal stromal cells (MSCs) is influenced by numerous epigenetic factors, among which microRNAs (miRNAs) play a key role. MiRNAs act as regulators of gene expression important for osteogenesis and help maintain skeletal tissue homeostasis. Numerous studies have demonstrated that dysregulation of specific miRNAs is not only associated with osteogenesis but also with the development of bone diseases such as osteoporosis. Osteoporosis represents one of the major public health challenges, affecting approximately 200 million people worldwide. In this master’s thesis, we studied the impact of selected miRNAs (hsa-miR-375-3p, hsa-miR-363-3p, hsa-miR-20b-5p, hsa-miR-133a-3p, and hsa-miR-31-3p) on the osteogenic differentiation of MSCs. The MSCs used as the cellular model for the study were prepared through lentiviral transduction, resulting in either overexpression or knockdown of the selected miRNAs. In these cells we analyzed the expression of key osteogenic markers (ALPL, BGLAP, COL1A1 and RUNX2), using quantitative real-time polymerase chain reaction. The results were quantified by relative quantification using the standard curve method and statistically evaluated using Student's t-test. These results were further compared with Von Kossa staining. Based on the results, we assessed the influence of miRNAs on the osteogenesis of MSCs. The results of the thesis confirm the hypothesis that altered miRNA expression significantly affects osteogenic differentiation of MSCs. Among the studied miRNAs, hsa-miR-363-3p showed the greatest potential as a biological marker and/or therapeutic target, as qPCR results showed statistically significant differences in the expression of osteogenic genes compared to control samples, and qPCR results were consistent with Von Kossa staining. Our results contribute to a better understanding of the role of miRNAs in osteogenesis and their potential in the diagnosis and treatment of osteoporosis. Despite the promising findings, the use of miRNAs in clinical practice remains a challenge due to their nonspecificity, instability, and lack of efficient delivery systems. Further research using clinical samples and in vivo models are necessary for better understanding and to enable the clinical implementation of miRNAs.

• Ključne besede: mesenchymal stem stromal cells, micro-RNA, teriparatide, osteogenesis, teriparatide, osteoporosis