Vpliv sprememb G-kvadrupleksa v promotorju gena TNFSF11 na njegovo aktivnost : enoviti magistrski študij farmacije

Borovnik, Rebeka (avtor), Marc, Janja (mentor), Lovšin, Nika (komentor)

Obnavljanje kostnega tkiva je uravnano z različnimi mehanizmi in dejavniki, med katerimi ima pomembno vlogo sistem RANK/RANKL/OPG. V primeru neustreznega uravnavanja tega sistema se lahko poveča izražanje proteina RANKL in s tem neposredno zveča nastajanje in aktivnost osteoklastov, kar poveča razgradnjo kostnine in lahko vodi v nastanek kostnih bolezni, npr. osteoporoze. Pri uravnavanju izražanja gena za protein RANKL (TNFSF11) ima pomembno vlogo promotorska regija, saj v svojem zapisu vsebuje regulatorne elemente. Ti omogočajo vezavo različnih transkripcijskih faktorjev in tako pospešijo ali zavrejo izražanje RANKL. V promotorski regiji gena TNFSF11 se nahaja tudi struktura, imenovana G-kvadrupleks, za katero je bilo že v številnih študijah ugotovljeno, da ima velik vpliv na izražanje različnih genov. Namen magistrske naloge je bil ugotoviti, kakšen je vpliv G-kvadrupleksa in sprememb v njegovem zaporedju na aktivacijo promotorske regije v genu za RANKL (TNFSF11). Dodatno smo želeli proučiti vpliv SNP-jev rs2296533 in rs9533157, ki se nahajata v bližini G-kvadrupleksa in ugotoviti, ali imajo tudi spremembe na tem mestu vpliv na aktivacijo promotorja. Za analizo vpliva G-kvadrupleksa na aktivnost promotorja gena TNFSF11 smo naredili mutacijsko analizo promotorske regije in izmerili aktivnost mutiranega in nemutiranega promotorja s pomočjo dvojnega luciferaznega testa v treh sesalskih celičnih linijah – HeLa, A549 in HOS. Pri delu smo uporabljali plazmidne vektorje pGL3, ki so vsebovali del zapisa promotorske regije gena TNFSF11. Z mutagenezo smo v promotorju gena TNFSF11 ustvarili mutacije na različnih delih G-kvadrupleksa, mutirali smo tudi polimorfna mesta rs2296533 in rs9533157. Pripravljene plazmidne vektorje smo nato s postopkom transfekcije vnesli v vse tri sesalske celične linije. Sledilo je merjenje njihovega vpliva na aktivacijo promotorske regije gena TNFSF11 s pomočjo dvojnega luciferaznega testa. Vnesene mutacije so povečale (RANGdel, RANG1T, RANG4T, RANG1234T ter posamezna SNP-ja rs2296533 in rs9533157) oz. zmanjšale aktivnost (RANG2713T in dvojna mutacija rs2296533, rs9533157) promotorja v sesalskih celičnih linijah HeLa in A549, v celicah HOS pa njihov vpliv ni bil tako izrazit. Naši rezultati so pokazali, da G-kvadrupleks z normalnim zaporedjem nukleotidov znižuje aktivnost promotorja in že najmanjša sprememba v zaporedju močno spremeni njegovo delovanje. Z analizo SNP-jev (rs2296533 in rs9533157) smo ugotovili, da tudi ta dva polimorfizma vplivata na aktivnost promotorja za RANKL. Naše ugotovitve potrjujejo, da je tudi tridimenzionalna struktura nukleotidnih zaporedij pomembna za aktivnost promotorja in lahko doprinese k boljšemu genetskemu odkrivanju posameznikov s povečano kostno razgradnjo oziroma s povečanim tveganjem za osteoporozo.

• Jezik: Slovenski jezik
• Ključne besede: polimorfizem posameznega nukleotida TNFSF11 G-kvadrupleks mutageneza RANKL
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [R. Borovnik]
• Leto izida: 2018
• Št. strani: VIII, 45 f., I-III f.pril.
• PID: 20.500.12556/RUL-121659
• UDK: 616.71-007.234:616-074(043.3)
• COBISS.SI-ID: 4544625
• Datum objave v RUL: 21.10.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The study of G-quadruplex changes in TNFSF11 gene promoter on its activity

Izvleček

Bone remodeling is regulated by various factors and mechanisms, among which the RANK/RANKL/OPG system plays an important role. In case of its inadequate regulation, the expression of RANKL protein may increase and therefore increases the formation and activity of osteoclasts. That increases bone degradation and can lead to the formation of bone diseases, e.g. osteoporosis. The expression of RANKL gene (TNFSF11) is regulated by its promoter region, since it contains various regulatory elements. Those enable binding of various transcription factors that inhibit or accelerate its expression. In promotor region of TNFSF11 gene there is also a structure called G-quadruplex. Various studies show, that it has a major influence on the expression of different genes. The purpose of this thesis was to determine the effect of G-quadruplex changes in the RANKL gene (TNFSF11) on activation of its promoter region. Additionally we wanted to analyze the influence of SNPs rs2296533 in rs9533157, located near G-quadruplex, and determine whether these changes affect activation of the promoter as well. In order to analyze the influence of G-quadruplex in the promoter region of TNFSF11 gene, we conducted mutation analysis of the promoter region and measured the promoter activity by dual luciferase test in three mammalian cell lines – HeLa, A549 and HOS. In our experiments we used plasmid vectors pGL3 that contained part of the TNFSF11 gene promoter region. With mutagenesis in TNFSF11 gene promoter, we created mutations in various parts of G-quadruplex as well as in polymorphic SNPs (rs2296533 and rs9533157). Prepared plasmids were then transfected into all three mammalian cell lines. Their influence on activation of the promoter region of TNFSF11 gene was measured by using a double luciferase test. All tested mutations in HeLa and A549 mammalian cell lines increased (RANGdel, RANG1T, RANG4T, RANG1234T and individual SNPs rs2296533 and rs9533157) or decreased (RANG2713T and a double mutation rs2296533, rs9533157) the activity of promoter. However, their effect was not so prominent in HOS cells. Our results have shown that G-quadruplex with a normal nucleotide sequence reduces promoter activity and even the slightest change in the sequence greatly changes its activity. With the analysis of SNPs (rs2296533 and rs9533157), we have found that these two polymorphisms also influence the activity of RANKL promoter. Our findings confirm that the three-dimensional structure of nucleotide sequences is also important for promoter activity and can contribute to better genetic detection of individuals with increased bone degradation or increased risk of osteoporosis.

• Ključne besede: osteoporosis TNFSF11 G-quadruplex SNP mutagenesis