3'-UTR单核苷酸多态性影响CYP8B1基因表达致胆囊胆固醇结石形成的机制研究

Sterol 12α-hydroxylase (CYP8B1) is the key enzyme for synthesis of cholic acid (CA), and responsible for the balance between formation of CA and chenodeoxycholic acid (CDCA). CA is regarded to promote bile flow, biliary cholesterol hypersecretion, and cholelithogenesis, since such stones are less likely to develop when there is a low ratio between CA and CDCA in bile. Suppression of CYP8B1 has, therefore, been suggested as a possible therapeutic strategy for prevention and dissolution of gallstones. In a previous study, we identified that single nucleotide polymorphism (SNP) rs3732860 in the 3'untranslated region (3'UTR) of CYP8B1 showed significant association to gallstone diseases (GD) in the two-stage association study. The carriers of the allele A of SNP rs3732860 had lower risk of GD compared with the carriers of the allele G. To assess the function of SNP rs3732860, we performed a clinical- and laboratory-based preliminary experiment. First, we compared the CYP8B1 mRNA levels in twelve patients with the different SNP rs3732860 genotypes. Second, we constructed an adenoviral vector carrying 3'UTR that includes the SNP rs3732860 and transfected it into cultured liver cells. The different CYP8B1 mRNA levels between different genotypes of SNP rs3732860 and different luciferase expression between 3'UTR and control were detected, respectively. It has been accepted that 3'UTR of genes may be responsible for posttranscriptional regulation of gene-expression and involved in mRNA stability. In addition, most human genes contain multiple poly(A)sites, which lead to different 3'UTRs with different binding to trans-acting factors. So we hypothesize that SNP rs3732860 may be correlated with different 3'UTRs and involves in expression activity of CYP8B1 gene, and leads to formation of gallstone(s). Therefore, to validate these hypotheses, we design the study as follows: First, to investigate the correlation between SNP rs3732860 and different 3'UTRs, then the expression levels of genes involved in bile acid metabolism and composition of bile acids to determine if SNP rs3732860 is functional and if it directly contributes to bile acid metabolism and stone formation. Second, to screen RNA binding protein( RBP) and microRNA(miRNA) that can regulate the expression of CYP8B1, and compare the difference of biding sites of RBP and miRNA in 3'UTR of CYP8B1 between different 3'UTRs. Finally, to evaluate the regulation of different 3'UTRs on CYP8B1 expression with luciferase assays. The study will help to understand the regulational mechanism of 3'UTR on CYP8B1 expression and allow to develop the targeted "small-molecule" drugs for the treatment and prevention of GD.

CYP8B1决定胆酸合成，参与胆囊结石形成。课题组前期发现CYP8B1基因3'-UTR单核苷酸多态rs3732860参与胆囊结石病(GD)发病并影响基因表达，其机制尚不清楚。已知3'-UTR和反式因子结合实现对基因表达的调节，而个体间存在3'-UTR长度多样性，和反式因子结合能力不同。因此推测rs3732860不同基因型间存在3'-UTR长度多样性，对CYP8B1表达调节能力不同，从而影响胆囊结石形成。本研究①检测rs3732860不同基因型间3'-UTR长度多样性和CYP8B1表达与GD发病差异；②筛选调节CYP8B1表达的反式因子（RBP和miRNA），序列比对分析不同长度3'-UTR调节基因表达的反式因子结合位点差异；③构建相应长度3'-UTR报告基因表达载体研究其对基因表达的调节能力。拟阐明3'-UTR影响CYP8B1基因表达的机制，为GD和胆汁酸代谢相关疾病的防治提供治疗靶点。

我国胆囊结石病发病率已达10%左右，严重危害人民群众身体健康。胆汁酸是维持胆固醇和磷脂处于溶解状态的重要溶剂，在胆囊胆固醇结石的形成中起关键作用。固醇12α-羟化酶（Sterol 12α-hydroxylase, CYP8B1）决定胆酸（Cholic acid, CA）的形成。有报道认为CYP8B1决定胆汁中CA和鹅去氧胆酸(chenodeoxycholic acid，CDCA)的比例，和胆囊结石形成相关。本研究组前期研究发现CYP8B1单核苷酸多态性rs3732860和胆囊结石形成相关。在此基础上，本课题检测rs3732860不同基因型间CYP8B1基因表达及其和胆汁酸成分、胆囊结石形成的关系；筛选对CYP8B1基因表达可能有调节作用的microRNAs，并验证；同时检测胆囊结石和正常对照间胆汁酸代谢基因的表达差异及其和胆汁酸成分间的关系。结果发现人CYP8B1虽然是CA合成的关键酶，但不是决定胆汁CA/CDCA比例的关键因素，肠肝循环可能是主要影响因素，胆汁酸重吸收FXR-OATP轴和胆汁中DCA升高有关，从而影响胆结石的形成。本研究在国内外首次发现FXR-OATP轴参与胆囊结石病的发生，将为胆囊结石病和胆汁酸代谢相关疾病的防治提供新的切入点。