miR-29c通过非经典模式调控酒精性肝损伤的作用机制研究

Alcohol abuse seriously endangers human health. It is very important to clarify the toxic mechanism of alcohol for the prevention and control of alcoholic liver injury. Previously, we found that the expression of miR-29c in the liver of alcoholic hepatitis patients was significantly down-regulated, suggesting that it plays an important role in the process of alcoholic-induced liver injury. To further clarify the biological toxicity effect caused by abnormal expression of miR-29c, a significant positive correlation was found between the expression of miR-29c and its target genes ADH6, CYP2C9 and CYP3A4 by integrating GEO and TCGA databases. This project intends to further analyze the positive regulatory mechanism of miR-29c by means of microRNA precipitation and protein mass spectrometry. We will investigate the effects of miR-29c on alcohol-induced biological toxic effects, such as elevation of alanine aminotransferase and lipid denaturation, in mice with liver-specific expression of miR-29c. And we will try to evaluate whether miR-29c could be used as a biomarker of alcoholic liver injury by using blood samples from patients with alcohol-induced liver injury. The research results of this project will provide a new epigenetic regulation mechanism for the toxic effects of alcohol, and provide a theoretical basis for discovering new biomarkers and targets for the prevention and treatment of alcoholic liver injury.

酒精滥用严重危害人类的身体健康，明确酒精的毒效应机制对酒精性肝损伤的防控至关重要。前期我们发现miR-29c在酒精性肝炎患者肝脏中的表达发生显著下调，提示其在酒精致肝损伤过程中起重要作用。为了进一步明确miR-29c表达异常所致生物学毒性效应，通过整合GEO和TCGA数据库，发现miR-29c与靶基因ADH6、CYP2C9和CYP3A4的表达呈显著正相关。本项目拟利用miRNA沉淀和蛋白质质谱检测等实验手段进一步解析miR-29c对靶基因的正向调控作用机制；通过构建肝特异性表达miR-29c的小鼠，研究miR-29c对酒精诱导的谷丙转氨酶升高和脂质变性等生物学毒性效应指标的影响；通过利用饮酒致肝损伤人群血液样本评估miR-29c能否作为酒精性肝损伤的生物标志。本项目的研究结果可为酒精的毒效应提供一种全新的表观遗传调控机制，为发掘新的酒精性肝损伤预防和治疗的生物标志物和靶点提供理论依据。

酒精脱氢酶（ADH）在酒精代谢和酒精毒性中发挥着重要作用，但目前我们对微小RNA介导的ADH基因簇的调控知之甚少。在本项目中申请人发现miR-29c通过靶向ADH6基因上的增强子元件从而激活ADH基因簇的转录。酒精性肝炎病人肝脏miR-29c表达显著下调。随后的生化和分子实验证据表明，miR-29c显著提高ADH6 mRNA和蛋白表达水平，而不影响ADH6转录本的稳定性。进一步的实验证据表明，外源miR-29c可以转移到细胞核中，以非经典方式结合ADH6基因上的增强子元件。萤光素酶报告基因实验和染色质免疫共沉淀数据表明，miR-29c激活了增强子的活性，并显著增加RNA聚合酶II在ADH1A、ADH1B、ADH1C、ADH4和ADH6基因启动子区的富集。最后，外源miR-29c显著促进ADH基因簇全体基因前体转录本和成熟转录本的表达。总之，我们的数据表明miR-29c可能是参与ADH基因簇激活的一种新的表观遗传调节因子。