微小RNA-24在肝纤维化中的作用及机制研究

Liver fibrosis is a common pathological process for chronic liver diseases, which can lead to liver cirrhosis and even liver failure. Therapeutic options for liver fibrosis are still limited. Some microRNAs (miRNAs) have previously been indicated to be related to liver fibrosis, however, it is largely unclear whether these changes are causes or consequences. The critical miRNAs for liver fibrosis is undetermined.. Our previous data based on microRNA arrays and quantitative reverse transcriptase-polymerase chain reactions have indicated that miRNA-24 was significantly decreased in liver fibrosis. In addition, overexpression of miRNA-24 promoted the proliferation of a hepatic stellate cell line, HSC-T6. Based on these results, we will determine the role of miRNA-24 in liver fibrosis through both gain-of-function and loss-of-function experiments in vitro and in vivo as well. In addition, the target genes of miRNA-24 in hepatic stellate cells and hepatocytes will be identified through gene arrays, bioinformatic analysis, luciferase reporter assays and Western Blot. . Our project will identify a critical miRNA responsible for liver fibrosis and also provide a novel therapeutic approach for liver fibrosis.

肝纤维化是多种慢性肝脏疾病共同的一个病理过程，其可以发展为肝硬化乃至肝衰竭，但是目前依旧缺乏可以有效防治肝纤维化的方法。既往有研究提示了一些微小RNA与肝纤维化的发生相关，但是没有真正鉴定出一个肝纤维化发生的关键微小RNA。我们前期的微小RNA芯片和荧光定量PCR研究提示微小RNA-24在小鼠肝纤维模型中显著降低，增加微小RNA-24可以促进肝星状细胞系增殖。本课题拟在此基础之上，结合肝星状细胞、肝细胞和动物整体水平的微小RNA-24功能缺失性实验和功能获得性实验，明确其与肝纤维的关系，并借助基因芯片、生物信息学分析、荧光素酶实验和免疫印迹法揭示微小RNA-24介导肝纤维化的分子基础。本课题将鉴定出一个肝纤维化的关键微小RNA，并开发出一种新的防治肝纤维化的方法。

肝纤维化是多种慢性肝病的终末阶段，晚期可进展至肝硬化，严重危害人类健康。本项目在小鼠肝纤维化模型中发现差异性表达的微小RNA-24（microRNA-24，miR-24），运用功能获得性和缺失性实验，探讨miR-24 对肝纤维化发生时肝星状细胞（HSC）主要生物学行为的影响。同时，对miR-24调控肝纤维化的分子机制进行探索，揭示肝纤维化的转录后调控网络。我们发现在TGF-β刺激大鼠HSC-T6的模型及小鼠肝纤维化模型中，miR-24的表达降低。上调miR-24 抑制HSC的细胞活力、EdU 阳性细胞比例、细胞周期G1期向S期转化、细胞迁移及TGF-β诱导的HSC活化，下调miR-24 具有相反的作用。靶基因预测、免疫印迹法、双荧光素酶报告基因实验以及功能逆转实验证实ALK4是miR-24抑制肝星状细胞激活、增殖的一个靶基因。此外，我们发现过表达miR-24抑制成年小鼠原代肝星状细胞的增殖和活化，抑制miR-24具有相反的作用。最后，我们发现与健康对照人群相比，肝硬化患者血浆中miR-24含量显著降低。总之，miR-24在肝纤维化中表达下降，过表达miR-24可以抑制HSC的增殖、迁移及活化，而抑制miR-24具有相反的作用，ALK4是miR-24调控HSC增殖及活化的一个靶基因。本项目说明了miR-24在肝纤维化发生中的作用，为肝纤维化的治疗提供了新的策略。