Ocena vpliva aktivacije in utišanja gena za glukokortikoidni receptor NR3C1 na izražanje gena za RANKL v modelu sesalskih celic

Zupan, Manca (author), Marc, Janja (mentor), Lovšin, Marija Nika (comentor)

Glukokortikoidi so steroidni hormoni, ki uravnavajo številne fiziološke procese v telesu, njihovi sintetični analogi pa se uporabljajo za zdravljenje različnih obolenj, kot so vnetja in avtoimunske bolezni. Kot zdravila so terapevtsko zelo učinkoviti, njihova pomanjkljivost pa so številni stranski učinki, med katere sodi tudi z glukokortikoidi povzročena osteoporoza (angl. glucocorticoid-induced osteoporosis, GIO). Glukokortikoidi namreč zmanjšajo kostno formacijo in povečajo razgradnjo kostnega tkiva, kar vodi v izgubo kostne mase, poveča pa se verjetnost za zlome. Ker molekularni mehanizmi, ki vplivajo na razvoj GIO, še niso v celoti pojasnjeni, smo se v raziskovalnem delu osredotočili na eno iz med glavnih regulatornih molekul kostnega metabolizma, na ligand receptorja za aktivacijo jedrnega dejavnika κB, RANKL. Le-ta je pomemben aktivator osteoklastogeneze in s tem kostne resorpcije, zato nas je zanimalo, ali morda in kako glukokortikoidni receptor NR3C1 vpliva na izražanje gena za RANKL. Vpliv NR3C1 smo želeli proučiti na nivoju transkripcije in v ta namen uporabili modela dveh sesalskih celičnih linij – celice pljučnega tumorja A549 in osteosarkomske celice HOS. V prvem delu smo v celice A549 s transfekcijo vnesli različne količine plazmidnega konstrukta z zapisom za glukokortikoidni receptor (pCMV-NR3C1) in vsakič izmerili nivo izražanja RANKL z metodo kvantitativne verižne reakcije s polimerazo (qPCR). Statistično značilno zmanjšanje ekspresije RANKL je povzročila transfekcija celic s 1500 ng plazmidnega vektorja glede na celice, transfecirane s praznim plazmidom, ter transfekcija celic s 375 ng, 750 ng in 1500 ng pCMV-NR3C1 glede na netretirane celice. V nadaljevanju smo delu celic v gojišče dodali še deksametazon, vendar statistično signifikantnih razlik v izražanju gena po dodatku tega liganda za receptor NR3C1 nismo zaznali. V drugem delu smo želeli preveriti vpliv utišanja gena za glukokortikoidni receptor. Uporabili smo obe celični liniji A549 in HOS in jih transfecirali s siRNA, ki je usmerjala nukleaze k razgradnji mRNA za protein NR3C1. Tudi tokrat smo izražanje RANKL izmerili z metodo qPCR, vendar statistično značilnih rezultatov nismo dobili. Naši rezultati so pokazali, da aktivacija glukokortikoidnega receptorja NR3C1 v celicah A549 zmanjša izražanje RANKL, kar je bilo v nasprotju z našimi pričakovanji in predhodnimi ugotovitvami, da glukokortikoidni receptor povečuje aktivnost promotorja gena za RANKL. Ker zastavljenih hipotez nismo mogli potrditi, bo potrebno narediti še več raziskav o glukokortikoidni regulaciji RANKL. Predvsem bi bilo smotrno določiti točna zaporedja vezavnih mest za glukokortikoidni receptor na RANKL in preučiti vpliv dimerizacije receptorja na aktivacijo ter na represijo promotorja RANKL. Vsa nova odkritja bi tako lahko prispevala k razvoju zdravilnih učinkovin za z glukokortikoidi povzročeno osteoporozo oz. k razvoju bolj varnih protivnetnih učinkovin, ki ne bi imele tako negativnih vplivov na kostno tkivo.

• Language: Slovenian
• Keywords: z glukokortikoidi povzročena osteoporoza, glukokortikoidni receptor NR3C1, ligand receptorja za aktivacijo jedrnega dejavnika κB (RANKL), kvantitativna verižna reakcija s polimerazo (qPCR)
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121021
• Publication date in RUL: 29.09.2020

Secondary language

• Language: English
• Title: Assessment of impact of glucocorticoid receptor NR3C1 gene activation and silencing on RANKL gene expression in mammalian cell models

Abstract

Glucocorticoids are steroid hormones which regulate numerous physiological processes in the human body, while their synthetic analogs are used to treat various diseases such as inflammation and autoimmune disorders. They are therapeutically highly effective as drugs, but their disadvantages are numerous side effects including glucocorticoid-induced osteoporosis (GIO). Glucocorticoids are inhibitors of bone formation and stimulators of bone resorption, which leads to loss of bone mass and increases the probability of fractures. Because molecular mechanisms influencing the development of GIO have not yet been fully explained our research was focused on one of the major regulatory molecules of bone metabolism – receptor activator of nuclear factor κB ligand, RANKL. This ligand is an important activator of osteoclastogenesis and as such an activator of bone resorption. Therefore, the main interest of our research was the impact of glucocorticoid receptor NR3C1 on RANKL gene expression on transcriptional level. For this purpose, we used two mammalian cell models: lung cancer cells A549 and cells of osteosarcoma HOS. In the first part of our research different quantities of plasmid construct containing the insert of glucocorticoid receptor (pCMV-NR3C1) were transfected into A549 cells and the level of RANKL expression was measured every time by quantitative polymerase chain reaction (qPCR). When we compared cells transfected with 1500 ng of plasmid with cells treated with empty vector and cells transfected with 375 ng, 750 ng or 1500 ng of pCMV-NR3C1 with untreated cells the results showed statistically significant inhibition of RANKL expression. Subsequently, dexamethasone was added to half of the cells but no statistical difference in gene expression was observed after the addition of this NR3C1 receptor ligand. In the second part of our research we wanted to examine the impact of silencing of glucocorticoid receptor gene on RANKL expression. Both A549 and HOS cell lines were used and transfected with siRNA, which recognizes and cleaves mRNA of NR3C1 protein. RANKL expression was measured by the qPCR method again but no statistically significant results were obtained. Our results showed that activation of the glucocorticoid receptor NR3C1 inhibits RANKL expression in A549 cells, which was contrary to our expectations and previous findings, according to which the glucocorticoid receptor increases the activity of the RANKL gene promoter. Since we have not been able to confirm our hypotheses, further research on the glucocorticoid regulation of the RANKL gene is necessary. In particular, it would be useful to determine the exact sequences of glucocorticoid receptor binding sites on RANKL and to examine the effect of receptor dimerization on RANKL promoter activation and repression. All new findings could contribute to the development of new treatments for glucocorticoid-induced osteoporosis or towards the development of safer anti-inflammatory drugs with less negative effects on bone tissue.

• Keywords: glucocorticoid-induced osteoporosis, glucocorticoid receptor NR3C1, receptor activator of nuclear factor κB ligand (RANKL), quantitative polymerase chain reaction (qPCR)