Ocena vpliva G-kvadrupleksa v promotorju gena TNFSF11 na njegovo aktivnost

Gošnjak, Teja (author), Lovšin, Marija Nika (mentor)

Osteoporoza je najpogostejša presnovna bolezen kostnine, za katero je značilno izguba kostne mase in propad kostnega tkiva. Posledično so kosti šibkejše in bolj nagnjene k zlomom. Vzrok za nastanek osteoporoze je neravnovesje med izgradnjo in razgradnjo kostnine. Proces kostne prenove je med drugimi uravnan preko sistema RANKL/RANK/OPG, ki nadzoruje proces osteoklastogeneze. V promotorski regiji gena TNFSF11 najdemo tudi strukturo imenovano G-kvadrupleks. G-kvadrupleksi so sekundarne prostorske strukture nukleinskih kislin, ki jih najdemo v zaporedjih, bogatih z gvanini. Narejenih je bilo že veliko študij, ki so pokazale, da ima struktura G-kvadrupleksa pomemben vpliv na izražanje številnih genov. Namen magistrske naloge je bil preučiti, ali spremembe v zaporedju G-kvadrupleksa promotorske regije gena TNFSF11 vplivajo na izražanje proteina med procesom prepisovanja ali med procesom prevajanja. To smo preverili tako, da smo v človeško celično linijo HeLa (celice raka materničnega vratu) vnesli reporterske plazmide s promotorsko regijo z G-kvadrupleksom. Pri delu smo uporabili reporterski plazmidni vektor pGL3 z delom zapisa promotorske regije gena TNFSF11, pri katerem smo v G-kvadrupleks vnesli zamenjavo baze gvanin v bazo timin na različnih mestih. Plazmide smo s transfekcijo vstavili v celice HeLa. Vpliv spremembe zaporedja G-kvadrupleksa na proces prepisovanja smo določili z analizo mRNA prepisane regije z metodo kvantitativne verižne reakcije s polimerazo (qPCR), vpliv na proces prevajanja pa smo določili z merjenjem aktivnosti promotorja gena TNFSF11 s testi luciferazne aktivnosti. Rezultate plazmidov s spremenjenim zaporedjem G-kvadrupleksa smo primerjali z rezultati izvornega plazmida. Na podlagi rezultatov smo ugotovili, da imajo vse testirane spremembe v G-kvadrupleksu promotorja gena TNFSF11 vpliv na izražanje proteina RANKL. Ugotovili smo tudi, da imajo spremembe večji vpliv na proces prevajanja kot na proces prepisovanja. Ne moremo pa zagotovo trditi, da imajo spremembe vpliv samo na enega izmed teh procesov, za to bi bile potrebne nadaljnje študije, s katerimi bi lahko bolj podrobno opazovali vpliv sprememb v G-kvadrupleksu na proces prepisovanja.

• Language: Slovenian
• Keywords: RANKL, TNFSF11, G-kvadrupleks, osteoporoza
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151841
• Publication date in RUL: 22.10.2023

Secondary language

• Language: English
• Title: The assessment of the G-quadruplex effect within the TNFSF11 promoter region on its activity

Abstract

Osteoporosis is the most common metabolic bone disease, characterized by the loss of bone mass and deterioration of bone tissue. Consequently, the bones become weaker and more prone to fractures. The cause of osteoporosis is an imbalance between bone formation and bone resorption. The process of bone remodeling is regulated, among others, by the RANKL/RANK/OPG system, which controls the osteoclastogenesis process. In the promoter region of the TNFSF11 gene, we also find a structure called G-quadruplex. G-quadruplexes are secondary spatial structures of nucleic acids that are found in sequences rich in guanine. Many studies have been conducted that have shown that the structure of the G-quadruplex has a significant impact on the expression of numerous genes. The purpose of this master's thesis was to examine whether changes in the sequence of the G-quadruplex in the promoter region of the TNFSF11 gene affect the expression of the protein during transcription or translation. This was tested by introducing reporter plasmids with the promoter region containing the G-quadruplex into the human cervical cancer cell line, HeLa. We used the reporter plasmid vector pGL3 with a portion of the promoter region of the TNFSF11 gene, in which we introduced a guanine to thymine base substitution in the G-quadruplex at various positions. The plasmids were transfected into HeLa cells. The impact of the G-quadruplex sequence change on transcription was determined by analyzing the mRNA transcript region using quantitative polymerase chain reaction (qPCR), and the impact on translation was determined by measuring the activity of the TNFSF11 gene promoter using luciferase assays. The results of plasmids with modified G-quadruplex sequences were compared to the results of the original plasmid. Based on the results, we found that all tested changes in the G-quadruplex of the TNFSF11 gene promoter have an impact on the expression of the RANKL protein. We have also found that changes have a greater impact on the translation process than on the transcription process. However, we cannot definitively claim that changes affect only one of these processes; further studies would be necessary to more closely observe the influence of changes in the G-quadruplex on the transcription process.

• Keywords: RANKL, TNFSF11, G-quadruplex, osteoporosis