Vpliv genetske variabilnosti in izražanja specifičnih mikroRNA na koncentracije PCSK9 med zdravljenjem z zaviralci PCSK9

Karun, Tina (author), Trebušak Podkrajšek, Katarina (mentor), Šebeštjen, Miran (comentor)

Protein proproteinske konvertaze subtilizin/keksin tipa 9 (PCSK9) pomembno posega v proces uravnavanja koncentracije holesterola LDL preko delovanja na receptorje za holesterol LDL na površini hepatocitov. PCSK9 se namreč veže na receptorje za holesterol LDL in celoten kompleks se razgradi v lizosomih in endosomih, zaradi česar na površini hepatocitov primanjkuje receptorjev za holesterol LDL in koncentracija holesterola LDL v krvi ostaja visoka. Medtem ko sta mehanizem delovanja PCSK9 in njegova vloga v celotnem procesu uravnavanja koncentracije holesterola LDL že dodobra proučena, pa njegovo uravnavanje s strani različnih dejavnikov še ni povsem raziskano. V sklopu magistrske naloge smo zato proučevali vpliv variabilnosti gena PCSK9 ter izražanja specifičnih mikroRNA na koncentracije PCSK9 pri bolnikih med zdravljenjem z zaviralci PCSK9, ki so preboleli zgodnji miokardni infarkt in imajo povišane koncentracije lipoproteina (a). Poleg 68 bolnikov smo v raziskavo vključili še 16 zdravih preiskovancev. Bolnike smo randomizirali v dve skupini. Prva skupina (N = 40) je šest mesecev prejemala zaviralec PCSK9, druga skupina (N = 28) pa šest mesecev placebo in nato šest mesecev zaviralec PCSK9. Bolnikom in zdravim preiskovancem smo določili ravni izbranih miRNA s kvantitativnim PCR ter genotipizirali izbrane genetske spremembe v genu PCSK9 s sondami TaqMan. Poleg tega smo v serumskih vzorcih določili koncentracije celotnega PCSK9 z encimsko imunoadsorpcijsko metodo. Pridobljeni rezultati potrjujejo vpletenost miR-483-5p v proces uravnavanja PCSK9 in vivo, saj smo z linearnim regresijskim modelom odkrili statistično značilen vpliv miR-483-5p na koncentracije PCSK9 pri bolnikih ob vključitvi v raziskavo. Pri preostalih proučevanih miRNA (miR-191-5p, miR-224-5p in miR-337-3p) vpliva na koncentracije PCSK9 nismo odkrili, kljub temu da obstajajo statistično značilne razlike v njihovem izražanju med bolniki in zdravimi preiskovanci ob vključitvi v raziskavo ter pri bolnikih med obdobjem zdravljenja z zaviralci PCSK9. Prav tako nismo odkrili vpliva proučevanih genetskih polimorfizmov na koncentracije PCSK9 pri bolnikih, kar je v nasprotju s številnimi do sedaj opravljenimi raziskavami. Razlogov za to je lahko več, bistvena pa sta predhodno zdravljenje s statini in prejemanje zaviralcev PCSK9. Omenjena zdravila namreč pomembno vplivajo na koncentracije analiziranega PCSK9, kar otežuje natančno oceno genetskega vpliva.

• Language: Slovenian
• Keywords: Protein proproteinske konvertaze subtilizin/keksin tipa 9, mikroRNA, genetski polimorfizem, genotipizacija, kvantitativni PCR.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158483
• Publication date in RUL: 14.06.2024

Secondary language

• Language: English
• Title: The effect of genetic variation and specific microRNA expression on PCSK9 concentrations during treatment with PCSK9 inhibitors

Abstract

The protein proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the regulation of LDL cholesterol concentration by binding to the LDL cholesterol receptors on the surface of hepatocytes. Following this extracellular binding of PCSK9 to the cell surface of the LDL cholesterol receptor, the complex undergoes lysomal and endosomal degradation, leading to lack of LDL cholesterol receptors on the surface of hepatocytes and subsequently to higher plasma LDL cholesterol levels. Although the mechanism of action and role of PCSK9 in the regulation of LDL cholesterol levels are well understood, the influence of other factors on this process is still not fully elucidated. Therefore we focused on the effects of genetic variations and specific microRNA expression on PCSK9 concentrations during treatment with PCSK9 inhibitors in patients who have suffered an early myocardial infarction and have very high lipoprotein (a) levels. A total of 68 patients and 16 healthy voluenteers were included in the study. We randomized the patients into two groups. The first group (N = 40) received PCSK9 inhibitor for six months, while the second group (N = 28) initially received a placebo for six months and then continued treatment with the PCSK9 inhibitors for further six months. Specific microRNAs were analysed in plasma samples from patients and healthy volunteers using quantitative PCR and selected PCSK9 gene variants were genotyped using Taqman assays. Additionally we measured total concentrations of PCSK9 in serum samples using an enzyme-linked immunosorbent assay. Using linear regression, we found a significant association between miR-483-5p expression and serum PCSK9 levels in patients at baseline. Our results therefore support a role for miR-483-5p in the regulation of circulating PCSK9 in vivo. While other microRNAs (miR-191-5p, miR-224-5p and miR-337-3p) were not significantly associated with PCSK9 levels, we still found significant changes in their expression in patients compared to healthy volunteers at baseline and in patients after six months of treatment with PCSK9 inhibitors. In contrast to other studies, we were also unable to detect any effects of genetic polymorphisms on PCSK9 concentrations. There could be several reasons for this, e.g. the previous treatment with statins and PCSK9 inhibitors. These drugs have a significant impact on PCSK9 levels, making it difficult to accurately assess the specific genetic effect.

• Keywords: Proprotein convertase subtilisin/kexin type 9 protein, microRNA, genetic polymorphism, genotyping, quantitative PCR.