## 3 Discussion
Of course, a direct effect of DNA methylation on transcriptional activity is expected, but the observed discrepancies between mRNA and protein (Table 2) could be due to different _SLC10A1_ transcripts in liver tissue that were not quantified in our study. As shown in Supplementary Figure S1, a significant correlation between mRNA and protein was observed, which might be even higher considering all _SLC10A1_ transcripts present in the liver. Moreover, other factors might influence mRNA expression and protein abundance. We found that the expression of the precursor miRNA MIR99 AHG was significantly correlated with NTCP expression (Figure 6A). This precursor miRNA, which is also affiliated to long intergenic non-protein coding RNA, contains the miR-99a/let-7c/miR-125b-2 cluster. Interestingly, MIR99 AHG was described recently as a tumor suppressor gene in lung adenocarcinoma [72] and was associated with advanced tumor progression and poorer prognosis in gastric cancer [73]. Moreover, MIR99 AHG served as an oncogene in acute megakaryoblastic leukemia [74]. In silico analysis suggested only for let-7c a poorly conserved binding site in the _SLC10A1_ 3'UTR using miRNAs target prediction tools (mirdb.org, targetscan.org, PolymiRTS). However, hepatic let-7c expression [75] did not correlate with NTCP expression, suggesting that the positive correlation of MIR99 AHG with NTCP expression is due to other indirect effects, which need further investigation. In addition, regulation by transcription factors might explain variability in mRNA expression. For instance, regulation by interleukin 1\(\beta\) via decreasing binding of HNF1\(\alpha\) to its promoter was described for mouse _SLC10a1_[76] and NRF2 has been identified as a transcriptional regulator of _SLC10A1_[77]. However, neither _HNF1\(\alpha\)_ nor _NRF2_ mRNA expression levels (determined in our transcriptome analyses) significantly correlated with either _SLC10A1_ mRNA expression or NTCP protein levels. In contrast, we observed a significant correlation between the constitutive androstane receptor (CAR; _NR1I3_) and _PPARA_ and _SLC10A1_/NTCP expression. The regulative role of PPARa in bile acid metabolism including hepatic NTCP expression changes upon PPARa activation has been shown in mice [78; 79]. However, these results are contradictory as for instance different activators of PPARa increased or decreased NTCP expression levels. Our data underlines the contribution of PPARa but suggests also CAR as well as PXR (_NR1I2_) and LXRa (_NR1H3_; \(\text{r}_{\text{S}}>0.35\), \(p<0.5\)) as direct or indirect regulators of NTCP expression in humans. The downregulation of NTCP associated with high CRP as a marker for inflammation mediated by cytokines is further supported by the negative correlation between STAT3 and _SLC10A1_ mRNA levels in our study since STAT3 regulates several genes involved in the inflammatory response [80]. In line with this, recently a STAT3-mediated downregulation of hepatic transporters including _SLC10A1_ has been shown in mice [81]. Generally, it can be assumed that NTCP has an influence on cellular hepatic metabolism because of its role in the uptake of important endogenous substances, like bile acids. However, NTCP also undergoes a complex post-translational regulation process mediated by metabolic signaling pathways which seem to be species-specific [1]. We, therefore, performed a gene set enrichment analysis using transcriptome data, which indicates that NTCP protein abundance is significantly associated with peroxisomal and fatty acid degradation, as well as bile acid biosynthesis pathways. The association with the peroxisomal pathway is supported by a study by [PERSON] et al. [82]. Here, NTCP protein levels were impaired in PEX2 mutant mouse livers. Generally, NTCP expression levels seem to be majorly associated with metabolic pathways in the liver. This observation is supported by a recent study by [PERSON] et al.