Vpliv mutacij v regiji G-kvadrupleksa promotorja gena TNFSF11 na njegovo aktivnost

Kovačič, Mateja (author), Lovšin, Marija Nika (mentor)

Vzrok za bolezni kosti je največkrat neravnovesje med razgradnjo in izgradnjo kostnine. Najbolj razširjena presnovna kostna bolezen je osteoporoza, ki lahko nastane zaradi motenj v dveh glavnih regulatornih mehanizmih. Prvi je sistem RANKL/RANK/OPG, ki je glavni pri nadzoru osteoklastogeneze, drugi pa je signalna pot Wnt, ki ima vlogo pri osteoblastogenezi. V magistrski nalogi smo se osredotočili na sistem RANKL/RANK/OPG. Pri neustreznem delovanju tega sistema lahko pride do povečanega izražanja proteina RANKL, ki se veže na membrano osteoklastov in poveča njihovo število ter aktivnost. Gen, ki kodira za protein RANKL, se imenuje TNFSF11. Promotorska regija gena TNFSF11 vsebuje regulatorne elemente in strukturo, imenovano G-kvadrupleks. Regulatorni elementi omogočajo vezavo različnih transkripcijskih dejavnikov, ki lahko pospešijo ali zavrejo izražanje gena TNFSF11. V magistrski nalogi smo proučevali, kakšen vpliv imajo spremembe v zaporedju G-kvadrupleksa na aktivnost promotorja gena TNFSF11. Pri delu smo rokovali s plazmidnim vektorjem pGL3, ki je vseboval del promotorske regije gena TNFSF11, v katerega smo vnesli mutacije na različnih mestih G-kvadrupleksa (RANG2713T, RANG2719T in RANG27T). Plazmide smo nato s transfekcijo vstavili v celice HeLa (celice raka materničnega vratu) in s pomočjo dvojnega luciferaznega testa izmerili vpliv na aktivacijo promotorja. Pri vseh pripravljenih plazmidih z vnesenimi spremembami je prišlo, glede na izvorni plazmid, do znižanja aktivnosti promotorja (pri mutacijah RANG2713T in RANG2719T se je aktivnost zmanjšala za faktor 0,2; pri RANG27T pa za faktor 0,4). Znižana aktivnost promotorja pa pomeni manjše izražanje proteina RANKL in posledično zmanjšano razgradnjo kosti. Proučevali smo tudi vpliv dveh genetskih sprememb v genu TNFSF11 – rs201748497 in rs200072190 – pri obeh smo zamenjali po en bazni par, kar je prav tako povzročilo znižanje aktivnost promotorja tega gena (pri rs201748497 se je aktivnost promotorja v primerjavi z izvornim plazmidom zmanjšala za faktor 0,9; pri rs200072190 pa za faktor 0,7). Iz dobljenih rezultatov lahko zaključimo, da vsaka sprememba, ki smo jo vnesli, vpliva na spremembo aktivnosti promotorja gena TNFSF11, posledično na izražanje proteina RANKL in s tem na nastanek osteoporoze. S tem smo doprinesli nove informacije o vplivu posameznih sprememb v genu TNFSF11, kar bi lahko pomagalo pri razvoju bolj učinkovitih in selektivnih učinkovin, ki bi se vezale bodisi na sam G-kvadrupleks ali kateri drugi sistem, ki sodeluje pri procesu kostne prenove (RANK/RANKL/OPG).

• Language: Slovenian
• Keywords: Osteoporoza, TNFSF11, RANKL, G-kvadrupleks
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-117103
• Publication date in RUL: 24.06.2020

Secondary language

• Language: English
• Title: The influence of mutations in the G-quadruplex region of TNFSF11 gene promoter on its activity

Abstract

The cause of bone disease is most often an imbalance between bone resorption and bone formation. The most common metabolic bone disease is osteoporosis, which can occur because of disorders in the two main regulatory mechanisms. The first one is the RANKL/RANK/OPG system, which is the most important in the control of osteoclastogenesis; and the second one is the Wnt signalling pathway, which plays a role in osteoblastogenesis. In this master thesis, we focused on the RANKL/RANK/OPG system. When this system is not working properly, it can result in increased expression of the RANKL protein, which binds to the membrane of osteoclasts, and can increase their number and activity. The gene encoding the RANKL protein is called TNFSF11. The promoter region of the gene TNFSF11 contains regulatory elements and a structure called G-quadruplex. The regulatory elements enable the binding of various transcription factors that can accelerate or inhibit the expression of the TNFSF11 gene. In this thesis, we studied the influence of changes in the G-quadruplex sequence on the activity of the TNFSF11 gene promoter. Using the plasmid pGL3 vector, which contained part of the TNFSF11 gene promoter region, we inserted mutations on different parts of the G-quadruplex (RANG2713T, RANG2719T and RANG27T). With transfection, we then introduced plasmids into HeLa cells (cervical cancer cells), and we measured the effect on the promoter activation using a double luciferase assay. In comparison to the original plasmid, all the prepared plasmids with mutations showed a decrease in the promoter activity (In the RANG2713T and RANG2719T mutations, the activity decreased by a factor of 0,2 and in RANG27T by a factor of 0,4). Decreased promoter activity means lower RANKL protein expression and consequently bone resorption. We also studied the influence of two genetic alterations in the TNFSF11 gene – rs201748497 and rs200072190, replacing one base pair in both, which also led to a decrease in this gene’s promoter activity (in comparison to the original plasmid, the promoter activity in rs201748497 decreased by a factor 0,9 and in rs200072190 by a factor 0,7). According to the obtained results, we can conclude that every alteration we introduced affects the change of the TNFSF11 gene promoter activity, consequently the protein RANKL expression, and thus the occurrence of osteoporosis. This provided new information on the impact of individual changes in the TNFSF11 gene; which could help to develop more effective and selective agents that would bind either to the G-quadruplex or any other system involved in the bone repair process (RANK/RANKL/OPG).

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