miRNA介导非酒精性脂肪肝CYP3A4和OATP1B1表达变化及药物代谢改变机制研究

Non-alcoholic fatty liver disease (NAFLD) is a kind of worldwide common chronic liver disease. Evidence from both clinical and animals studies has shown that the expression and activity of the major drug metabolism enzyme cytochrome P450 3A (CYP3A) and the organic anion transporting polypeptides (OATPs) family of drug transporters are decreased in NAFLD.The exact mechanisms for these decreases remain unknown at large, and more clinical investigations are needed. MicroRNAs (miRNAs) are a group of non-coding RNAs that negatively modulate the expression of target genes at translational level. The expression of several miRNAs possibly targeting CYP3A4 or OATP1B1 directly is reported to be elevated in NAFLD. In combination of clinical study using probe drugs as a tool with cellular and animal studies by using molecular techniques such as semi-quantitative real time PCR, Western blot and RNA interference, this project is designed to investigate the effects of NAFLD on pharmacokinetics of probe drugs and confirm the role of miRNAs in regulating the expression of CYP3A4 and OATP1B1 in the liver in NAFLD patients.

非酒精性脂肪性肝病（NAFLD）是常见的慢性肝病。基于人肝微粒和实验动物的研究均发现，NAFLD时肝脏主要药物代谢酶CYP3A及肝细胞表面摄取型转运体蛋白的表达和活性均显著下降，但具体机制不明，且缺乏人体内试验研究。miRNA是一类新的非编码小分子RNA，在翻译水平负性调控靶基因的表达；NAFLD患者肝脏以CYP3A4和OATP1B1为潜在靶基因的多种miRNA表达发生明显改变。本项目拟采用代谢酶探针药物为工具进行临床试验的基础上，同时结合细胞和动物实验，运用实时定量PCR、Western blot和RNA干扰等分子生物学实验方法，明确NAFLD对药物代谢动力学的影响，查明相关miRNA在NAFLD时CYP3A4和OATP1B1表达下调中的作用，首次从miRNA介导的表观遗传学角度，探讨NAFLD时体内CYP3A4和OATP1B1活性下降的分子机制，为指导临床合理用药提供理论依据和奠定基础

非酒精性脂肪性肝病（NAFLD）是常见的慢性肝病，它影响着肝脏主要药物代谢酶CYP3A及肝细胞药物转运体蛋白的表达和活性。在此病理过程中，miRNA作为一类内源性的具有广泛调控功能的非编码RNA，可产生相应作用；本项目使用游离脂肪酸（FFA）诱导张氏肝细胞建立NAFLD细胞模型，运用实时定量PCR和Western blot等分子生物学实验方法，测定了模型细胞中miRNA-194,miR-511,miR-150，miR-200a，以及CYP3A4，OATP1B1的表达情况。结果表明，在模型细胞中，miR-511表达上调；miR-150，miR-200a显著上调；miRNA-194显著下调；CYP3A4和OATP1B1的mRNA和蛋白表达均下调。继而，通过双荧光素酶报告基因和RNA干扰的途径，我们查明了相关miRNA在NAFLD导致CYP3A4和OATP1B1表达下调过程中所产生的作用。我们认为：NAFLD病理条件极可能通过miR-150和miR-200a介导CYP3A4下调，而miR-511虽可靶向SLCO1B1 mRNA，并使OATP1B1下调，但是否介导NAFLD下调OATP1B1，仍有待研究。另外，miRNA-194对OATP1B1无此类影响。本研究首次从miRNA介导的表观遗传学角度，探讨NAFLD影响体内CYP3A4和OATP1B1表达的分子机制，为指导临床合理用药提供理论依据和奠定基础。