环状RNA hsa circ_0060477/miRNA/AKT3环路调控自噬抑制HSC活化

Previous evidences suggested autophagy of hepatic stellate cell (HSC) promoted its activation. However, it is not well known that whether circular RNA (circRNA) plays an important role in the autophagy and activation of HSC. In our recent study, hsa circ_0060477, a circRNA regulated by HNF4α, was shown down-regulated in activated HSC. The computational algorithm analysis revealed that circ_0060477 might sponge miR-646, miR-503-5p and miR-6838-5p to regulate their common target gene Akt3 expression. In this study, we will further focus on the role of circ_0060477/miRNA/ AKT3 circuit in HSC autophagy and activation. First of all, the association of differential expression of hsa circ_0060477 and hepatic fibrogenesis will be determined. Second, hsa circ_0060477 will be up-regulated or repressed in HSC to investigate the effect of this circRNA on its direct target miRNA, as well as Akt3 expression and HSC autophagy, which might lead to the significant regulatory effect on HSC activation and hepatic fibrosis. Then we will demonstrate the crosstalk between HNF4α, circ_0060477, miR-646, miR-503-5p, miR-6838-5p and Akt3 and illuminate the molecular mechanism of hsa circ_0060477/miRNA/AKT3 circuit in HSC activation. Finally, the inhibitory effect of circ_0060477 on hepatic fibrosis will be investigated in vivo, which may represent a promising novel therapeutic method for hepatic fibrosis.

既往研究证实HSC自噬可促进其活化，尚无环状RNA (circRNA)调控HSC自噬及活化的相关研究。前期发现，hsa circ_0060477在活化HSC中显著下调，且受HNF4α调控。它可能和miR-646、miR-503-5p、miR-6838-5p及上述miRNA共同靶基因Akt3形成环路抑制HSC自噬及活化。本课题进一步证实hsa circ_0060477差异表达与肝纤维化的相关性；在HSC内增加或阻遏hsa circ_0060477表达，研究其竞争抑制相关miRNA、调控Akt3的作用，观察其对HSC自噬、活化及肝纤维化的影响；阐明hsa circ_0060477与相关miRNA、Akt3及HNF4α的“crosstalk”，研究hsa circ_0060477/miRNA/Akt3环路调控HSC自噬和活化的分子机制；并在体内探讨hsa circ_0060477的抗纤维化作用。

既往研究证实HSC自噬可促进其活化，尚无环状RNA (circRNA)调控HSC自噬及活化的相关研究。前期发现，hsa circ_0060477在活化HSC中显著下调，且受HNF4α调控。它可能和miR-646、miR-503-5p、miR-6838-5p及上述miRNA共同靶基因Akt3形成环路抑制HSC自噬及活化。本研究中我们利用实时PCR技术发现在HSC活化、大鼠实验性肝纤维化加重及人肝硬化过程中后hsa_circ_0060477表达下调，miR-646、miR-503-5p、miR-6838-5p上调、HNF4α下调，Akt3表达下调；证实hsa_circ_0060477差异表达与肝纤维化相关。生物信息学分析及双萤虫素酶报告基因系统证实hsa_circ_0060477可与miR-646、miR-503-5p、miR-6838-5p竞争结合，而Akt3是miR-646、miR-503-5p、miR-6838-5p沉默的共同靶基因。成功构建过表达hsa_circ_0060477的腺病毒Ad-hsa_circ_0060477和抑制hsa_circ_0060477的siRNA- hsa_circ_0060477。将Ad-hsa_circ_0060477感染活化的人HSC细胞株LX-2，证实上调hsa_circ_0060477阻断HSC活化、抑制部分HSC自噬基因表达；利用siRNA下调hsa_circ_0060477表达，发现下调hsa_circ_0060477促进HSC活化、激活部分HSC自噬基因表达。通过回补实验证实hsa_circ_0060477主要靶向miR-646并调控其下游基因Akt3，参与HSC增殖活化及自噬的调节；ChIP assay表明HNF4α通过结合hsa_circ_0060477基因启动子DNA对hsa_circ_0060477的转录发挥调控作用。构建硫代乙酰胺（TAA）致肝纤维化大鼠模型，利用腺病毒载体在体内上调hsa_circ_0060477，发现hsa_circ_0060477具有体内抗肝纤维化作用。通过该研究，培养硕士研究生1名，博士研究生1名。发表2篇SCI论文， 1篇被接收。