miRNA对UGT1A1表达和活性的调控及机理研究

UDP-glucuronosyltransferase (UGT) 1A1 is an important drug phase II enzyme. It is responsible for gulcuronidation of many human endogenous signalling molecules, drugs and carcinogens. miRNAs are short (~22 nt long) endogenous noncoding RNAs that act on a partially complementary segment within the 3'-untranslated region (3'-UTR) of target transcript, leading to translation inhibition or mRNA cleavage. Because miRNAs play important roles in regulating the metabolic activity of drug-metabolism enzymes and influence the curative effect and toxicity of drugs though the regulation of their expression, they becaome current research hotspot. Based on previous knowledge, we start with miRNAs screening which can target on UGT1A1, and the regulation of miRNAs on these two enzymes in in normal colon cells and colon carcinoma, then their effect on the cytotoxicity of irinotecan which is often used to treat metastatic colorectal cancer. Our subject is to indentify the role of miRNAs involed in UGT1A1 regulation and the activity of anti-tumor drug. By investigating the relationship between miRNAs and UGT1A1, we will provide new mechanism information for individual administration and clinical rational drug use.

UGT1A1是一种重要的二相代谢酶，能催化许多内源性分子和药物的代谢。miRNA则是长度为22 nt左右的内源非编码小RNA，通过作用于相应靶mRNA的3'端非编码区导致靶基因的降解或翻译抑制。由于miRNA能够通过调控药物代谢酶的表达来改变其代谢活性，从而影响药物疗效或毒性，因此成为目前的研究热点。本课题在前期工作的基础上，拟通过分子生物学方法在结肠癌细胞中筛选出能够与UGT1A1作用的miRNA；进而在正常结肠细胞和结肠癌细胞中，研究UGT1A1的表达和抗肿瘤药物伊立替康、尼洛替尼的细胞毒性的关系；阐明miRNA对UGT1A1的调控作用机理和对抗肿瘤药物活性的影响，明确miRNA与UGT1A1甲基化、UGT1A1表达和抗肿瘤药物活性的关系,为指导UGT1A1底物个体化用药提供科学依据。

尿苷二磷酸葡萄糖醛酸转移酶（UGT1A1）是一种重要的二相代谢酶，能催化许多内源性物质和药物的代谢。其表达受多种因素影响，比如miRNA和DNA甲基化等。了解UGT1A1异常表达机制，可为指导UGT1A1底物个体化用药提供科学依据。我们应用软件预测了与UGT1A1相互作用的miRNA，通过报告基因实验筛选，结果表明miR-200a和miR-141与UGT1A1 3’端非编码区作用较强，并呈现浓度依赖性。检测到UGT1A 3'-UTR存在3个SNP位点，且MAF值均大于10%。报告基因功能筛选结果表明，miR-1286可以与SNP(rs8330)位点结合，可能抑制UGT1A1的转录或翻译。与配对的癌旁组织相比，在肝和结肠癌组织中UGT1A1、1A3和1A4的mRNA水平均显著下调；在结肠癌组织中UGT1A8、1A9和1A10的表达量也显著低于配对的癌旁组织。在肝癌组织中miR-200a-5p的表达量显著升高，miR-200a-3p和miR-143-3p有微弱上调，而miR-141-3p和miR-183-5p的表达水平无显著变化；在结肠癌组织中，只有miR-183-5p的表达量显著升高，其他4个miRNA的表达水平则都没有显著变化。miRNA转染细胞后表达量显著增加，但没有显著性抑制UGT1A1的转录和翻译。以伊立替康的代谢物SN-38为底物，检测miRNA对细胞毒性有影响。利用DNA甲基化转移酶1（DNMT1）抑制剂地西他滨（Decitabine，简称DAC）和Zebularine（ZEB）处理细胞，可以诱导HepG2和HT29细胞中UGT1A1表达。miR-148a和miR152在肝癌组织中的表达显著下调，转染HepG2和HT29细胞，DNMT1表达没有差异。报告基因实验证明，miR-148a和miR-152没有直接与UGT1A 3'-UTR相互作用，而可能通过抑制DNMT表达，影响UGT1A1启动子区甲基化，最终改变UGT1A1的表达水平。