Analiza diferencialnega izražanja genov v človeških osteosarkomskih celicah divjega tipa in z izbitim genom FUBP3

Milostnik, Nina (author), Lovšin, Marija Nika (mentor)

Študije asociacije na celotnem genomu (GWAS študije) se uporabljajo za odkrivanje različic na genomu, pri katerih je statistično tveganje za pojavnost določene bolezni višje. Rezultati teh študij pričajo o SNP polimorfizmu v genu FUBP3, ki bi lahko vplival na kostno remodelacijo in bil povezan z nižjo mineralno kostno gostoto in povečanim tveganjem za zlome. Na podlagi rezultatov GWAS študij smo torej FUBP3 opredelili kot potencialno tarčo, ki bi lahko sodelovala v procesih kostnega metabolizma in vplivala na mineralno kostno gostoto. Cilj magistrske naloge je ovrednotiti vpliv gena FUBP3 na kostni metabolizem, saj o njegovem vplivu na tem področju ni še nič znanega. Hipoteze smo postavili na podlagi rezultatov, predhodno pridobljenih na Katedri za klinično biokemijo Fakultete za farmacijo UL. Ugotovljeno je bilo namreč, da je bila v celicah, ki jim je bil gen FUBP3 izbit, motena mineralizacija. Gen FUBP3 smo funkcijsko ovrednotili s pomočjo rezultatov analize diferencialnega izražanja genov MMP-1, VCAM-1, ALPL, CARMIL2, EDNRA, NFATC2, CHI3L1, MCAM in TUBB3. V okviru magistrske naloge smo s pomočjo metode kvantitativne verižne reakcije s polimerazo v realnem času v vzorcu, ki vsebuje človeške osteosarkomske celice, določili nivo izražanja izbranih genov. Nato smo določili nivo izražanja istih genov v celicah, ki jim je bil predhodno izbit gen FUBP3. Ugotavljali smo, kako se koncentracija posamičnega gena v celicah, v katerih je FUBP3 izbit, spremeni v primerjavi s celicami divjega tipa. Na podlagi tega rezultata in v literaturi pridobljenih informacij o tem, kako analizirani geni vplivajo na kostni metabolizem, smo iskali potencialne mehanizme, prek katerih bi lahko FUBP3 vplival na mineralno kostno gostoto. Po interpretaciji rezultatov smo hipoteze, ki smo jih postavili na podlagi rezultatov, pridobljenih na Fakulteti za farmacijo, potrdili, saj se je pri večini rezultatov pokazalo, da je nivo izražanja gena FUBP3 sorazmeren z mineralno kostno gostoto in nanjo vpliva pozitivno, odkrili smo pa tudi potencialne signalne poti, prek katerih bi FUBP3 lahko vplival na mineralno kostno gostoto.

• Language: Slovenian
• Keywords: Kostni metabolizem, osteoporoza, osteogena diferenciacija, FUBP3, GWAS.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-160576
• Publication date in RUL: 31.08.2024

Secondary language

• Language: English
• Title: Differential gene expression analysis in human FUBP3 gene knockout versus wild-type osteosarcoma cells

Abstract

Genome-wide association studies (GWAS studies) are being used for discovering genome variants that are associated with statistically higher incidence of certain diseases. In GWAS studies SNP polymorphism that could be associated with bone remodelation, lower mineral bone density and higher risk of bone fracture was identified in FUBP3 gene. Based on GWAS results we have defined FUBP3 as a potential target that could participate in bone metabolism processes and influence mineral bone density. The purpose of this master thesis is to functionally evaluate the impact of FUBP3 gene on bone metabolism since its role in this field is unknown. Our hypothesis were set based on results obtained on University of Ljubljana Faculty of Pharmacy's Department of Clinical Biochemistry. These studies have established tha bone mineralization is impaired in FUBP3 KO cells. FUBP3 was functionally evaluated based on the results of differential gene expression analysis results. We have analysed genes MMP-1, VCAM-1, ALPL, CARMIL2, EDNRA, NFATC2, CHI3L1, MCAM and TUBB3. In this master thesis we determined level of expression of choosen genes in samples containing human osteosarcoma cells by using real-time polymerase chain reaction method. After that we determined level of expression of the same genes in FUBP3 knock out cells. We compared concentration of each gene in FUBP3 knock out cells to its concentration in wild-type cells. Based on this result and the sources and literature obtained information on how the analysed genes affect bone metabolism, we searched for potential mechanisms involved in FUBP3-related mineral bone density changes. After the interpretation of our results we have accepted our proposed hypothesis, based on results previously obtained on Faculty of Pharmacy since most results have shown that FUBP3 gene is proportional with mineral bone density and affect it positively. We have also discovered some potential signal pathways that could be involved in FUBP3-related mineral bone density changes.

• Keywords: Bone metabolism, osteoporosis, osteogenic differentiation, FUBP3, GWAS.