供体NR1I3基因多态性影响肝移植术后他克莫司代谢个体差异的机制研究

Our previous study found that donor NR1I3 gene polymorphism were associated with tacrolimus concentration/dose (C/D) ratios after liver transplantation, the results demonstrate that NR1I3 gene may affect metabolism of TAC. However, the gene functional loci of NR1I3 recently remains unclear.CYP3A5 gene polymorphisms were associated with tacrolimus C/D ratios, but the polymorphisms could not fully explain individual difference. Literature show that the CAR of NR1I3 gene expression can specifically combine ER-6 motif of CYP3A5 gene promoter, then promotes the expression of CYP3A5. Therefore, we speculate that there is a progressive mechanism called "functional loci of NR1I3 gene→NR1I3 gene expression→activity of CYP3A5 enzyme→TAC metabolism" after liver transplantation. The study intends to use sequencing technology, bioinformatics analysis, et al to determine the functional loci in NR1I3 gene. Then buildling nonlinear mixed-effect model and establishing different liver cell lines using site-directed mutagenesis to compare the CYP3A5 and NRI13 expression to validate the functional loci. Finally, through liver transplant model in rats to validate that donor NR1I3 affects the metabolism of tacrolimus. We aim to provide new markers for the personalized medicine of tacrolimus.

课题组前期研究发现供体NR1I3基因多态性与肝移植术后他克莫司（TAC）C/D值相关，提示NR1I3基因影响TAC代谢。然而，其功能位点目前尚不明确。药物代谢酶CYP3A5基因多态性与TAC C/D值相关，但不能完全解释TAC代谢个体差异。文献报道NR1I3基因表达产物CAR可特异性结合CYP3A5启动子区ER-6 motif，促进CYP3A5表达。据此，我们推测移植后供肝内存在“NR1I3功能位点→NR1I3表达→CYP3A5酶活性→TAC代谢”逐层影响机制。为证实以上假设，本课题拟采用测序技术、生物信息学方法等明确NR1I3基因的功能位点；利用非线性混合效应模型和定点突变技术比较肝细胞株NR1I3、CYP3A5基因表达差异验证此功能位点；通过大鼠肝移植模型验证供体来源的NR1I3基因通过调控CYP3A5基因表达影响TAC代谢，旨在为TAC个体化用药提供新的遗传标志物。

课题组前期研究发现供体NR1I3基因多态性与肝移植术后他克莫司（TAC）C/D值相关，提示NR1I3基因影响TAC代谢。然而，其功能位点目前尚不明确。我们利用大临床肝移植样本回顾性分析NR1I3与肝移植术后他克莫司代谢的关系，发现NR1I3 rs11265572和rs11584174遗传多态性与肝移植术后FK506代谢表型相关联。通过体内外试验我们观察到NR1I3 rs11584174 AG/GG供肝在肝移植术后早期肝细胞损伤后应激状态时NFκB通路活性高，致使PXR转录活性降低，下游基因CYP3A5酶表达下调，从而导致FK506慢代谢，表现为高C/D值。进一步我们还完成了肝移植术后早期他克莫司供受体CYP3A5 rs776746+受体CYP3A4 rs2242480, SLC28A3 rs7853758, SLCO1B1 rs2291075 +供体 FMO3 rs1800822快-中-慢代谢新分型，拟进一步建立数学模型指导临床用药。此外，我们还发现NR1I3和Kruppel样因子6（KLF6）与肝肝癌复发相关。进一步研究发现KLF6通过影响下游PCNA和MMP-9水平，抑制肝癌细胞的生长和侵袭。