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Holesterol je verjetno najbolje poznani steroid, saj kot prekurzor igra pomembne biokemijske vloge, hkrati pa je ključen za strukturo in delovanje membran vretenčarjev. Zaradi pomembnih funkcij holesterola je vzdrževanje njegove homeostaze bistvenega pomena. V raziskavi smo želeli dobiti vpogled v povezavo prekinjene sinteze holesterola in cirkadianega izražanja genov signalnih poti Hedgehog (HH) in Wingless (WNT). Na celičnih linijah HepG2 z izbitimi posameznimi geni za zapis encimov pozne poti sinteze holesterola (CYP51, DHCR24, HSD17B7, SC5D) smo s qPCR kvantificirali izražanje genov, ki so del omenjenih signalnih poti. Pri 10 od 15 genov smo izmerili diferenčno izraženost pri vsaj enem genotipu z izbitim genom. To nakazuje, da lahko prekinjena sinteza holesterola neposredno ali posredno vpliva na signalizaciji HH in WNT. Za potrditev hipoteze, da ima vpliv tudi na cirkadiano izražanje teh genov, smo na celičnih linijah z izbitimi geni izvedli cirkadiani poskus. Potrdili smo ciklično izražanje gena DBP, ki je izhodni gen cirkadiane ure in pogosto uporabljana pozitivna kontrola. Nivo izražanja smo ovrednotili še za komponenti signalizacije WNT, DKK4 in LEF1. Rezultati so pokazali, da LEF1 ne oscilira, smo pa pri celicah z izbitim genom CYP51 morda odkrili zmožnost lanosterola in dihidrolanosterola za zagon cirkadiane ure gena LEF1. Pri genu DKK4 smo poleg cikličnega izražanja opazili tudi povečano amplitudo in izražanje gena pri vseh linijah z izbitimi geni glede na nativno. Spremembe v cirkadiani fazi izražanja nismo uspeli potrditi pri nobenem od izmerjenih genov signalizacije WNT. Cholesterol is probably the best-known steroid, as it plays important biochemical roles as a precursor and is at the same time crucial for the structure and function of vertebrate cell membranes. Given its important functions, maintenance of its homeostasis is essential. We have studied the connection between disrupted cholesterol synthesis and circadian expression of Hedgehog (HH) and Wingless (WNT) signalling pathways on human HepG2 cell lines with different gene knockouts (KO) (CYP51, DHCR24, HSD17B7, SC5D) from the late part of cholesterol synthesis. We quantified the expression level for selected genes on qPCR, where 11 out of 15 turned out to be differentially expressed in at least one KO genotype. The latter indicates that disrupted cholesterol synthesis modulate studied signalling pathways, directly or indirectly. To confirm our hypothesis that circadian expression of selected genes is also affected, we performed a circadian experiment on CYP51, DHCR24 in SC5D KO. We confirmed the cyclic expression of the DBP gene, which is circadian clock output gene and a frequently used positive control. We also measured the expression level for DKK4 and LEF1, which are important components of the WNT signalling. The results showed that LEF1 gene does not cycle, although we might discovered the ability of lanosterol and dihydrolanosterol to start the circadian clock of LEF1 in CYP51 KO. In addition to cyclic expression of the DKK4 gene, significantly increased amplitude and expression was observed in all KO lines. Circadian expression of selected genes showed no indication of changes in circadian parameters. |
