Dicer1基因及其调控的miRNA在肝再生中的作用研究

Micro RNAs (miRNAs) participate in various physiopathological processes of the liver, including development, metabolism and tumorigenesis, depending on its role in the regulation of target genes. Some of the miRNAs are also confirmed to participate in the regulation of liver regeneration. It has been found more than 400 miRNAs in the liver, however, their roles are still unclear. Generally, one miRNA targets a group of genes, and one gene might be regulated by several miRNAs, therefore, the functional overlaps of the miRNAs make it difficult to clarify the actual function of one miRNA using gene-knockout animal. To systemically investigate the role of miRNAs in the liver, the Dicer 1 gene, which catalyzes the maturation of pre-miRNA, was knock outed using Cre-Loxp system. However, the early deficiency of the Dicer 1 gene results in disruption of liver development and function after birth, making this mouse unsuitable for liver regeneration study. In this study, we will establish an Alb Cre-ERTs/Dicer 1loxp/loxp mouse which Cre recombinase is driven by albumin promoter and induced by tamoxifen. Using this mouse strain, we can knock out the Dicer 1 gene in mature hepatocytes before partial hepatectomy. We will explore the effect of the loss of Dicer 1 gene and the subsequent deficiency of miRNAs on liver regeneration. Furthermore, we are aimed to identify several miRNAs which closely involved in liver regeneration and rescued their expression in hepatocyte. Finally, we will investigate the role of re-expressed miRNAs in liver regeneration and wish to identify some crucial miRNAs which could significantly promote the liver regeneration.

小RNA（miRNA）通过调控特定靶基因表达发挥其功能，与多种肝脏病理生理活动相关；一些miRNA也被证实参与了肝再生调控。肝脏内miRNA数量众多，彼此间有功能交叉，某个miRNA的缺失往往可由其他成员代偿，故基于单个miRNA敲除模型来探讨其功能的研究存在缺陷。Dicer 1是催化miRNA成熟的关键酶，敲除其基因将导致绝大多数miRNA缺失。报道显示在胚胎期特异敲除肝脏Dicer 1基因后，小鼠肝细胞发育和功能受到严重影响，并不适用于研究Dicer 1及其miRNA产物在肝再生中的作用。本研究将建立可诱导的肝细胞特异Dicer 1基因敲除小鼠模型，仅在围手术期敲除其成熟肝细胞Dicer 1基因。我们将观察Dicer 1及miRNA缺失对发育成熟肝脏的再生、代谢的影响；探讨维持肝脏正常功能和再生的重要miRNA及其机制；以期寻找到调节肝再生的关键miRNA。

本国家自然科学基金项目以“基因敲除----再生差异----microRNA回输”为研究主线及特色，以Dicer1基因敲除小鼠为模型，行70%肝切除后与野生型小鼠对照，探究microRNA与肝脏再生的关系,并通过回输差异microRNA的类似物，再次比较验证再生情况。在课题实施过程中，遵循设计方向及创新思想，并根据实验结果调整研究方法，研究步骤为：1.采用Alb-Cre/Loxp重组系统，通过Dicerflox/flox小鼠与Albumin-cre工具鼠杂交，得到肝细胞特异性敲除Dicer1小鼠。2. 建立条件性Dicer1基因敲除小鼠及野生型小鼠2/3肝切除模型，分别于术后各时间点采集血液和肝脏标本，检测肝细胞再生情况、肝再生相关基因蛋白表达水平、microRNA表达水平及肝功能和脂代谢指标；3.根据实验结果通过回输microRNA21的类似物，再次检测肝再生情况。在研究实施过程中，实验进行顺利，研究成果如下：1.通过Dicerflox/flox小鼠与Albumin-cre工具鼠杂交可建立肝细胞特异性敲除Dicer1小鼠，为研究Dicer1基因提供了理想的动物模型。2.发现Dicer1基因相关的microRNA能够促进肝细胞快速进入增殖过程，加速肝再生进程。肝再生早期，基因敲除小鼠肝再生受到抑制，进入细胞周期缓慢。3. 通过Dicer1敲除小鼠中的AKT信号通路抑制分子高表达，确定 microRNA21的靶基因，通过回输microRNA21的类似物，检测肝再生情况可以基本抵消由Dicer1缺失引起的肝再生抑制。本研究发现及验证了microRNA21对肝脏再生的重要作用，对 Dicer1的作用提出新的思考。为通过基因敲除小鼠研究肝再生提供了新的思路，并证明了“基因敲除----再生差异----microRNA回输”研究思路的可行性。本课题收集、保存的肝移植供体血浆标本及临床资料仍可为后续研究提供样本资料。