一个新的lncRNA通过miR145调控肝星状细胞活化促进肝纤维化的分子机制

Hepatic stellate cells (HSCs) play a key role in liver fibrosis/cirrhosis. Recently, noncoding RNA have emerged as pivotal regulators in the activation of HSCs. Our team has investigated the role of small noncoding RNA—microRNA145(miR 145) in the activation of HSCs and liver fibrosis and has identified that Krupple like factor 4 (KLF4) is the direct target gene of miR 145. However, the role of long noncoding RNA in the activation of HSCs and liver fibrosis remains unclear. We have identified a new lncRNA HSCs activation relative factor (lncRNA HARF), which is upregulated in activated HSCs. Moreover, bioinformatics analysis suggests a potential correlation between lncRNA HARF and miR145. Overexpression of lncRNA HARF could increase miR145 level in HSCs. The aim of study is to explore the role of lncRNA HARF in liver fibrosis/cirrhosis and the underlying molecular mechanisms by lncRNA HARF to modify the biogenesis of miR145. In this study we will exam the expression of lncRNA HARF in fibrotic liver tissues from both cirrhotic patients and rat models induced by CCl4. To reveal the underlying mechanism, we will further investigate the precise pathway of lncRNA HARF to regulate biogenesis of miR145 including direct and indirect ways. More importantly, the therapeutic role of lncRNA HARF or miR145 knock down will also be investigated to pave the way for new antifibrotic therapies. Taken together, this study might contribute to increase our knowledge of the mechanisms underlying HSCs activation and might be beneficial to future clinical diagnosis and therapy for hepatic fibrosis and liver cirrhosis.

肝星状细胞（HSCs）活化是肝纤维化发生的关键环节，非编码RNA参与调控了HSCs活化。本课题组前期已证实miR145可通过KLF4-TGFβ通路促进HSCs活化；并鉴定了一个新的与HSCs活化相关的长链非编码RNA即lncRNA HARF。生物信息学分析发现HARF可能是miR145前体，过表达HARF可上调miR145表达。我们提出“lncRNA HARF是一种新的HSCs活化调控因子，其通过miR145参与HSCs活化促进肝纤维化”假设。为此，拟重点研究大鼠原代HSCs、大鼠肝纤维化模型与肝纤维化/肝硬化患者肝组织中HARF、miR145表达；lncRNA HARF调控miR145的机制；利用维A耦联及超声微泡技术靶向干预肝纤维化大鼠HSCs中HARF及miR145的表达，并观察其肝纤维化程度的变化，以证实上述假设。藉此，丰富和完善肝纤维化的发病机制理论并为其治疗提供全新靶点。

肝纤维化发生的中心环节是肝星状细胞（hepatic stellate cells, HSCs）的活化。近来报道非编码RNA（ncRNA）在HSCs活化及肝纤维化过程中发挥了重要作用，可能成为肝纤维化治疗的潜在靶点。本课题组前期通过实验证实了miR145可通过KLF4-TGFβ通路促进HSCs活化；并鉴定了一个新的与HSCs活化相关的长链非编码RNA（lncRNA）。基于此前研究，我们提出“lncRNA 是一种新的HSCs活化调控因子，其通过调控miRNA参与HSCs活化促进肝纤维化”的假设。因此，在国家自然科学基金面上项目的资助下，本项目继续通过Illumina高通量测序并验证筛选与HSCs活化相关的新lncRNA及miRNA，发现lncRNA NONRATT029085.2、miR-145、miR-199a-3p和miR-214等均表达上调，miR-30c和miR-142-3p表达下调。利用siRNA调控HSC-T6中lncRNA NONRATT029085.2表达，发现lncRNA NONRATT029085.2下调后HSCs活化受到抑制，这一过程伴随着fractalkine和CX3CR1表达下调，相关机制还在进一步研究。此外，抑制miR-145、miR-199a-3p和miR-214表达后，HSCs活化也明显降低。采用antagomiR-214和antagomiR-199a-3p注射肝纤维化小鼠后，其肝纤维化程度及纤维化指标明显下降。上述研究表明，lncRNA和miRNA可通过调控HSCs活化从而参与肝纤维化过程。本项目在国家自然科学基金支持下，我们团队已经在American Journal of Gastroenterology，Cell Death and Disease，Scientific Reports等SCI杂志发表论文16篇（第一标注11篇），培养博士研究生3名，硕士研究生3名，较好的完成了本项目的预期目标。