## 2 Results
### Genome-Wide Expression Correlation Analysis and Gene Set Enrichment Analysis  
In order to identify genes and pathways that are associated with NTCP expression, transcriptome and genes set enrichment analyses were performed using 118 liver tissues from our cohort with available genome-wide expression data. 1% and 0.1% of analyzed probesets comprising genes, pseudogenes and precursor microRNAs (\(n\) = 32,232) were significantly correlated (absolute rs \(>\) 0.04) to _SLC10A1_ mRNA expression (measured by qPCR) and NTCP protein levels, respectively. Then, we investigated whether pharmacogenes (\(n\) = 344) [63], target genes (\(n\) = 564) [64] or precursor miRNAs (\(n\) = 1372) were correlated with NTCP expression. The significantly correlated genes (adjusted \(p\) < 0.05, absolute rs \(>\) 0.4) predominately belonged to the pharmacogene group (_SLC10A1_ mRNA = 5.81%; NTCP protein = 1.45%; Figure 6A). Compared to the other groups, this overrepresentation was significant (\(p\) < 0.05). Both _SLC10A1_ mRNA expression and NTCP protein levels were significantly positively correlated with ALDH7A1 expression (_SLC10A1_: rs = 0.45; \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{3}{c}{_SLC10A1_ mRNA} & \multicolumn{3}{c}{**NTCP Protein**} \\ \cline{2-5}
**CpG\_site** & **Correlation** & **Unadjusted P** & **Correlation** & **Unadjusted P** \\ \hline
1\_CpG\_1 & 0.09 & 0.28 & 0.13 & 0.14 \\  
\hline \hline \end{tabular}  
Significant associations (without correction for multiple testing) are marked in gray. \({}^{\rm 6}\) CpG sites still significant after adjustment for multiple testing using Benjamini-Hochberg procedure.  
\end{table} 
Table 2: Correlation of DNA methylation at individual CpG sites with mRNA or protein levels of _SLC10A1_/NTCP. NTCP: \(\mathrm{r_{S}}=0.5\)), whereas highest negative correlation was found with ATP-binding cassette subfamily B member 2 (TAP1, \(\mathrm{r_{S}}=-0.52\)). Interestingly, also transcription factors and nuclear receptors correlated to NTCP protein levels (e.g., _NR1I3_ [CAR] \(\mathrm{r_{S}}=0.44\)), _PPARA_ (\(\mathrm{r_{S}}=0.44\)), _STAT3_\(\mathrm{r_{S}}=-0.45\)). Within the group of precursor miRNAs the highest significant correlation was found for MIR99 AHG (\(\mathrm{r_{S}}=0.41\)). Figure 6: Genome-wide expression correlation analysis and gene set enrichment analysis. (**A**) Violin plots showing correlation results between genome-wide expression of the selected gene groups and _SLC10A1_ expression levels as determined by qPCR (light blue) and NTCP protein levels (yellow),  
respectively. Top correlated genes (\(\text{r}_{\text{S}}\leq-0.4\) or \(\text{r}_{\text{S}}\geq 0.4\)) are labeled with gene names. Barplots of the right figure are showing the percentage of significantly correlated genes (adjusted \(p<0.05\) and \(\text{r}_{\text{S}}\leq-0.4\) or \(\text{r}_{\text{S}}\geq 0.4\)). Values are stratified by selected gene groups in both plot types. (**B**) KEGG pathway enrichment analysis of correlation results of _SLC10A1_ mRNA and NTCP protein levels using R package pathfindR. The gradient color reflects the lowest enrichment p value. Colored polygons indicate gene sets summarized using hierarchical clustering. The two top clusters are shown for _SLC10A1_ and NTCP protein. The peroxisome pathway is among the top clusters for both, _SLC10A1_ and NTCP protein. Overlap of the top 10 enriched terms for mRNA and protein are shown in the Venn diagram. (**C**) Heatmap visualizes the genes that are involved in the seven overlapping enriched pathways. The color gradient shows the Spearman correlation estimates. We next selected gene signatures from the Kyoto encyclopedia of genes and genomes (KEGG), gene ontology (GO) database, and metabolic signatures published in a study by [PERSON] et al. [65]. Gene set enrichment analyses were performed and revealed metabolic pathways associated with _SLC10A1_ mRNA expression and NTCP protein levels. In Figure 6B the two top clusters for enriched pathways are shown for _SLC10A1_ mRNA and NTCP protein.