## 5 Discussion
However, according to the ENCODE Transcription Factor Targets database, _HNF4a_ can act as a _CYP4F2_ transcription factor too. _HNF4a_ binding sites were identified in the promoter of _CYP4F2_, but no functional studies have been performed yet [47]. These results raised the hypothesis that hsa-miR-24-3p may indirectly affect _CYP4F2_ expression by acting through the transcription factor _HNF4a_. PXR and CAR are regulated by _HNF4a_[48; 49]. In turn/additionally, we hypothesized that _CYP4F2_ expression in extra-hepatic cells might also be controlled by PXR or CAR transcription factors. Detailed studies of miR-24-3p function in humans have not been conducted, but its association with CVD is well known. It has been shown that miR-24-3p inhibits angiogenesis in the myocardial infarction zone. Decreased survival, proliferation and migration of cells and angiogenic properties have been observed after the transfection of endothelial cells with this miRNA [50; 51]. Increased miR-24 expression inhibits the expression of endothelial inflammatory factor _TNF-a_, which contributes to the regulation of inflammation via the NF-kB signaling pathway. It is worth noting that patients with atherosclerosis had significantly lower miR-24 expression in their blood samples compared to healthy patients [50]. According to our previously published data, miR-24-3p expression in blood plasma was 4.4-fold higher in patients with stable angina than in healthy controls [33]. In the current study, transfection with the hsa-miR-24-3p mimic after 24 h of incubation resulted in a 1.7-fold decrease in _CYP4F2_ expression in HUVECs compared to the negative control. However, after transfection with the hsa-miR-24-3p mimic, the expression of _HNF4a_, _PXR_ and _CAR_ was not detected, suggesting that these transcription factors are not involved in the regulation of CYP genes in endothelial cells and that other regulatory mechanisms might possibly be involved. The evaluation of glucocorticoid-induced _HNF4a_ transcriptional activity was performed by applying treatment with 1 uM dexamethasone to HUVECs. _HNF4a_, _PXR_ and _CAR_ expression could not be determined. However, treatment with dexamethasone resulted in a change in _CYP4F2_ expression; dexamethasone downregulated the expression of _CYP4F2_ 1.96-fold. Dexamethasone was identified as a classical inducer of cytochrome P450 subfamily enzymes (CYP1A, CYP2B, CYP3A and CYP4A), but there are no data available on how dexamethasone acts on the CYP4F subfamily or CYP4F2 gene [52]. An analysis of CYP4F2 protein quantity was performed by ELISA to determine the effect of both the new-generation anticoagulant microarrayaban and hsa-miR-24-3p at the protein level. Transfection with hsa-miR-24-3p in HUVECs resulted in a 6.4-fold decrease in CYP4F2 protein levels in cells. However, exposure to rivaroxaban at different concentrations did not significantly alter CYP4F2 protein levels in HUVECs. The expression of the altered gene at the mRNA level may change the amount of the corresponding protein in the cell, as gene expression at the mRNA and protein levels often varies disproportionately. The same mRNA molecule can be used for the synthesis of several hundred to several hundred thousand protein molecules, and various post-translational mechanisms contribute to this [53]. The assessment of rivaroxaban metabolism in vitro using HUVEC culture as a model system could be seen as a limitation of the experiment. Most similar studies conductedpreviously cultivated cell cultures using two-dimensional methods that do not always mimic the environment of cells in vivo [54]. The metabolic profiling of rivaroxaban in HUVECs may differ from live organisms or three-dimensional cultures that have the advantage of cell-to-cell interactions.