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.
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