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.