Vpliv izbitja gena FUBP3 in prekomernega izražanja gena fubp3 cebrice v človeških osteosarkomskih celicah na njihovo osteogeno diferenciacijo

Gojko, Klara (author), Lovšin, Marija Nika (mentor)

Asociacijske študije v celotnem genomu (GWAS) so študije, ki obravnavajo številne pogoste genetske različice pri različnih osebkih, da bi ugotovili potencialne povezave z boleznimi. Ko so odkrite nove genetske povezave, lahko raziskovalci informacije uporabijo za razvoj boljših strategij za odkrivanje, zdravljenje in preprečevanje bolezni. GWAS študije so do zdaj podale veliko genskih lokusov povezanih s kostnimi boleznimi. Eden izmed odkritih lokusov je enonukleotidni polimorfizem rs7851693 v intronu gena za FUBP3, ki je povezan s tveganjem za zlome kosti oz. osteoporozo. Vloga proteina FUBP3 pri metabolizmu kosti še ni znana. V želji, da bi prispevali k raziskavam, smo poskušali določiti povezavo med izražanjem gena FUBP3 in kostnim rakom (osteosarkomom). V okviru magistrske naloge, smo najprej poskušali ugotoviti ali gen FUBP3 igra vlogo pri tvorbi kosti z uporabo tehnike popolnega izbitja gena med osteogeno diferenciacijo človeških osteosarkomskih celic (HOS, angl. human osteosarcoma cells ). Potek mineralizacije smo paralelno primerjali s potekom mineralizacije pri divjem tipu HOS celic. Z metodo barvanja Alizarin Red S in končnim kvantitativnim vrednotenjem smo ugotovili, da mineralizacija hitreje poteka pri divjem tipu, kar nakazuje, da gen FUBP3 ima določeno vlogo pri tvorbi kosti. Hitrejša mineralizacija je bila vidna tudi pod mikroskopom. Poleg tega smo naredili transfekcijo človeških HOS celic, z izbitim FUBP3 genom, s proteinom FUBP3 ribe zebrice (zfFUBP3) in praznim vektorjem (pCMV), da bi preverili ali povišano izražanje zfFUBP3 vpliva na osteogeno diferenciacijo. Rezultate smo tudi kvantitativno ovrednotili in ugotovili, da ima zfFUBP3 vpliv na tvorbo kosti in lahko sklepamo, da igra podobno vlogo kot človeški FUBP3. Poleg opisanih eksperimentov, smo z metodo PCR v realnem času (qPCR) kvantitativno izmerili še izražanje treh osteoblastnih genov – kolagena tipa I (COL1A1), osteokalcina (OC) in transkripcijskega faktora II (RUNX2). Rezultati magistrske naloge kažejo, da igra protein FUBP3 pomembno vlogo pri mineralizaciji osteosarkomske celične linije HOS.

• Language: Slovenian
• Keywords: FUBP3, osteosarkom, osteogena diferenciacija, qPCR
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-144713
• Publication date in RUL: 09.03.2023

Secondary language

• Language: English
• Title: The effect of FUBP3 gene knockout and zebrafish fubp3 gene overexpression in human osteosarcoma cells on their osteogenic differentiation

Abstract

Genome-wide association studies (GWAS) are studies that look at many common genetic variants in different individuals to identify potential associations with diseases. When new genetic associations are identified, researchers can use the information to develop better strategies for detecting, treating and preventing diseases. GWAS studies have so far identified many gene loci associated with bone diseases. One of the discovered loci is the single – nucleotide polymorphism rs7851693 in the intron of the FUBP3 gene, which is associated with the risk of bone fractures and osteoporosis. The role of the FUBP3 protein in bone metabolism is not yet known. In order to contribute to research, we tried to determine the relationship between the expression of the FUBP3 gene and bone cancer (osteosarcoma). As a part of the master’s thesis, we tried to determine whether the FUBP3 gene plays a role in bone formation using the technique of complete gene knockout during osteogenic differentiation of human osteosarcoma cells (HOS). The course of mineralization was compared in parallel with the course of mineralization in wild-type HOS cells. Using the Alizarin Red S staining method and the final quantitative evaluation, we found out that mineralization occurs faster in the wild-type cells, which suggests the FUBP3 gene has a certain role in bone formation. Faster mineralization was also visible under the microscope. In addition, we transfected human FUBP3 gene knocked out cells, with zfFUBP3 (zebrafish FUBP3) and empty plasmid, in order to check whether increased expression of this protein has an impact on osteogenic differentiation. We also evaluated the results quantitatively and found that zfFUBP3 has an impact in bone formation and we can conclude that it plays a similar role to human FUBP3. In addition to the described experiments, we quantitatively measured the expression of three more genes – collagen type I (COL1A1), osteocalcin (OC) and transcription factor II (RUNX2) – using the real-time PCR (qPCR) method. The results of the master's thesis show that the protein FUBP3 plays an important role in the mineralization of the osteosarcoma cell line HOS.

• Keywords: FUBP3, osteosarcoma, osteogenic differentiation, qPCR