长链非编码RNA竞争抑制miR-21调控肝纤维化的机制研究

Hepatic fibrosis represents the wound healing response to liver injury from a wide variety of etiologies. Long noncoding RNA (lncRNA) has been shown to play important role in the regulation of signaling pathways during hepatic fibrogenesis, which is the new area on the investigation of the mechanisms of hepatic fibrosis. In our previous study, differential expression of lncRNA AK123790 and uc002pyc has been observed in fibrotic liver compared to the normal control, which might be involved in the regulation of extracellular signal-regulated kinase (ERK) pathway in activated of hepatic stellate cell (HSC) and the epithelial-to-mesenchymal transition (EMT) process in hepatocyte due to their targeting repression of miR-21 expression. In this study, we will further confirm the changed level of lncRNA AK123790 and uc002pyc in liver tissues from fibrotic patients or rats using lncRNA microarray. Luciferase activity determination of the lncRNA reporter co-transfected with miR-21 mimic will be used to reveal the direct inhibitory effect of the lncRNA AK123790 or uc002pyc targeting miR-21 expression. Subsequently, the adenovirus vectors carried lncRNA AK123790 or uc002pyc will be constructed respectively to up-regulating the expression of the lncRNAs in the hepatocyte developed with EMT or activated HSC, which has been considered as the vital contributors to promoting hepatic fibrosis, then we would demonstrate the suppressive effect of the increasing expression of the lncRNAs on miR-21 level, the activation of ERK signal pathway and EMT process, as well as the attenuation of hepatic fibrogenesis. Additionally, we would also reveal the crosstalk between the lncRNA AK123790 or uc002pyc and miR-21, which might lead to the significantly divert expression of down-stream genes. Finally, after the adenoviruses which could up-regulate the expression AK123790 or uc002pyc are injected into the fibrotic liver of the experimental rats through the tail vein, respectively, the effect and mechanism of forced expression of lncRNA AK123790 or uc002pyc on the repression of hepatic fibrosis would be indicated, which might provide the new target for therapy of hepatic fibrosis.

肝纤维化是各种慢性肝病进展的必经阶段，lncRNA对信号通路的转录后调控是肝纤维化发病机制的研究热点。前期工作发现，lncRNA AK123790及uc002pyc与miR-21关系密切并在肝纤维化进程中存在差异表达，对肝星状细胞(HSC)ERK通路和肝细胞EMT进程可能同时有调控作用。本课题将利用lncRNA芯片鉴定纤维化肝脏lncRNA AK123790和uc002pyc差异表达；荧光报告基因证实lncRNA对miR-21的直接抑制作用；上调发生EMT的肝细胞及活化HSC中lncRNA表达，观察其对miR-21、EMT和ERK通路相关基因的抑制作用。调控miR-21的表达，检测lncRNA及下游基因表达，明确lncRNA与miR-21间"crosstalk"的作用。上调肝纤维化大鼠上述lncRNA表达，研究lncRNA对肝纤维化进程的抑制作用及分子机制，为肝纤维化治疗提供新的靶点。

lncRNA对信号通路的转录后调控是肝纤维化发病机制的研究热点。前期工作发现，lncRNA与miRNA关系密切并在肝纤维化进程中存在差异表达，对炎症信号通路和EMT可能同时具有调控作用。本研究基本按计划进行。首先利用lncRNA芯片及实时PCR检测证实人和动物纤维化肝脏存在lncRNA AK123790、uc002pyc、uc77表达下调及uc.420-表达上调；荧光报告基因证实lncRNA AK123790、uc002pyc、uc77对miR-21、uc.420-对miR-194的直接抑制作用。利用siRNA及过表达质粒分别或联合调控HSC-T6及原代HSC中lncRNA uc.420-及miR-194表达，可通过调控JAK-STAT通路活性及EMT改变HSC增殖活化及凋亡。研究还发现，过表达lncRNA AK123790和 uc77可降低miR-21水平，抑制HSC增殖活化及生物学活性；利用siRNA沉默lncRNA AK123790和 uc77则促进HSC增殖活化。上述研究结果证实，lncRNA 可竞争结合相关miRNA，调控炎症通路及EMT，参与肝纤维化进程。lncRNA可能成为肝纤维化治疗的新靶点。通过该研究，发表SCI收录论著1篇，核心期刊论文1篇，培养硕士研究生2名，博士研究生1名。