INTRODUCTION
The P-glycoprotein (P-gp) transporter, which is encoded by theABCB1 transporter gene (also known as the MDR1 gene), is widely distributed in intestinal epithelial cells, liver cells, and renal proximal tubule epithelial cells1. Previous studies have shown that ABCB1 may affect tacrolimus absorption, distribution, and excretion 2, 3. Thus, some studies have focused on evaluating the relationship between ABCB1 gene polymorphism (1236C>T, rs1128503, Gly412Gly; 677G>T/A, rs2032582, Ala893DSer/Thr; 3435C>T, rs1045642, Ile1145Ile of exons 12, 21, and 26) and tacrolimus pharmacokinetics3, 4. However, the findings of these studies are inconsistent as concerns both SNPs and haplotypes, and systematic mechanistic studies to support their conclusions are still lacking. In addition, recent studies carried out on ABCB1 have shown that it functions mainly in prolonging drug retention time in intestinal cells by pumping drugs into the intestinal lumen i.e. on the surface of the gastrointestinal tract, and this also affects tacrolimus absorption5-7. However, the effects of ABCB1 on tacrolimus metabolism in liver cells have not been clearly elucidated.
Although CYP450 and transporter (eg. ABCB1 ) gene polymorphisms are important for individual variations in drug metabolism and pharmacodynamics, they do not fully explain such individual differences8, 9. In a previous study [in publishing progress] , we recruited 78 patients with significantly different initial tacrolimus blood concentrations (C0> 10 μg/L or < 5 μg/L) following liver transplantation at the First Affiliated Hospital of Zhengzhou University, and corresponding donor liver samples were collected for the genotyping of CYP3A5 and other genes which have been reported in the literature to be possibly related to tacrolimus metabolism(Tables S1 and S2) . We found significant individual differences in tacrolimus plasma concentrations in CYP3A5 non-expressors (CYP3A5*3/*3 ), indicating that other key factors may also affect tacrolimus metabolism (Figure S1, S2) . In recent times, the influence of epigenetic factors on drug metabolism has received increasing research attention; among these factors, DNA methylation has become a new research hotspot. DNA methylation can alter gene expression through the external regulatory pathway without altering the primary structure of DNA, thereby affecting the metabolism of drugs and endogenous substances10. More specifically, cytosine on the CpG island combines with a methyl group transferred by DNA methyltransferase (DNMT) to produce methyl cytosine, which then inhibits DNA transcription 11. Previous studies have shown that DNA methylation is an important epigenetic factor that affects CYP450 gene expression12, 13. In a previous study, we also found DNA methylation to play an important role in CYP3A4transcriptional regulation14. In addition, anti-tumour drugs, such as daunorubicin, activate ABCB1 transcription by hypomethylating its promoter region, and this possibly results in multi-drug resistance15, 16.
Therefore, for this study, we selected the liver tissues of 23 donors carrying the CYP3A5*3/*3 genotype and exhibiting varied initial tacrolimus blood concentrations. DNA methylation sequencing was performed to screen for the different methylation sites of drug metabolism enzymes or drug transporters (such as ABCB1 ), and then we evaluated the relationship between the different methylation sites and the tacrolimus C0/D ratio. In addition, by treating HepG2 cells with methylase inhibitors, we further verified whether DNA methylation is a key epigenetic factor that affects tacrolimus metabolism.