长链非编码RNA lnc uc.420-竞争结合miRNAs调控HSC自噬及肝纤维化

Hepatic stellate cell (HSC) activation is a central mechanism underlying liver fibrogenesis. Recent investigations have established the role of autophagy as an important driver of HSC activation and hepatic fibrosis, and screening the important mediators and signaling pathways involved in autophagy of HSC might provide potential target for anti-fibrotic therapy. In our previous work, significant up-regulation of long noncoding RNA (lncRNA) lnc uc.420- has been observed in the fibrotic liver tissues from patients or experimental rats as well as in activated HSC, along with the decreased expression of miR-194 and miR -19b and enhanced level of down-stream gene autophagy-related gene 5 (Atg5), which was targeted by both of miR-194 and miR -19b. The computational algorithm analysis and luciferase reporter assay of lncRNA revealed that lnc uc.420- could sponge both of miR-194 and miR -19b to regulate Atg5 expression. In the current study, we will further focus on the association of differential expression of lnc uc.420-, miR-194, miR -19b and Atg5 with HSC activation and hepatic fibrogenesis. Then we would change lnc uc.420- level in HSC to investigate the effect on direct sponging miR-194, miR -19b, as well as Atg5 expression and HSC autophagy by lnc uc.420-, which might lead to the significant regulatory effect on HSC activation and hepatic fibrosis. At the same time, we would demonstrate the crosstalk between lnc uc.420-, miR-194, miR -19b and the activity of Atg5 and autophagy related signaling pathway. Finally, the inhibitory effect and mechanism of down-regulation of lnc uc.420- on hepatic fibrosis would be explored in fibrotic models, which would demonstrate the important role of lnc uc.420- in therapy of hepatic fibrosis.

HSC活化是肝纤维化发展的重要环节，HSC自噬增加可促进其活化，研究调控HSC自噬的分子及信号通路意义重大。前期工作发现，lnc uc.420-在纤维化肝脏及活化HSC中明显上调，其竞争结合的miR-194、miR -19b表达下调，miR-194和miR -19b共同靶基因自噬相关基因5（Atg5）表达上调。本课题进一步证实lnc uc.420-、miR-194、miR-19b、Atg5差异表达与肝纤维化的相关性；调控HSC内lnc uc.420-表达，研究其对miR-194、miR-19b竞争抑制、Atg5表达和HSC自噬、活化及肝纤维化进程的影响；阐明lnc uc.420-与miR-194、miR-19b、Atg5的“crosstalk”作用，明确lnc uc.420-对HSC自噬信号通路和活化的调控机制；探讨下调lnc uc.420-表达对肝纤维化的抑制作用，为治疗提供新的靶点。

肝星状细胞（HSC）活化是肝纤维化发展的重要环节，HSC自噬增加可促进其活化，研究调控HSC自噬的分子及信号通路意义重大。前期工作发现，lnc uc.420-在纤维化肝脏及活化HSC中明显上调，其竞争结合的miR-194-5p表达下调，miR-194-5p靶基因自噬相关基因5（Atg5）表达上调。本研究利用实时PCR技术在肝硬化组织样本、肝纤维化大鼠模型及大鼠HSC活化过程中证实存在lnc uc.420-、Atg5上调，miR-194-5p下调。生物信息学分析及双萤虫素酶报告基因证实lnc uc.420可竞争结合 miR-194-5p；而miR-194-5p靶向结合ATG5 3’-UTR。利用慢病毒上调HSC中lnc uc.420，miR-194-5p表达下调，Atg5上调，自噬相关指标及胶原表达相关指标升高，HSC凋亡减少，增殖增加。利用慢病毒下调HSC中lnc uc.420，miR-194-5p表达上调，HSC凋亡增加，增殖抑制，迁移能力降低，自噬减弱；肝纤维化相关指标α-SMA、ColⅢ、TIMP-2表达下调，MMP-2、MMP-9表达上调；自噬相关指标Atg5、Atg7、Baclin 1、P62下调，LC3a/b上调。调控miR-194-5p可部分逆转上述lnc uc.420对HSC活化及自噬的影响。体内利用慢病毒抑制lnc uc.420可延缓肝纤维化进程。上述研究结果证实，lnc uc.420-可竞争结合miR-194-5p，靶向Atg5，调控HSC自噬及活化，参与肝纤维化进程，抑制lnc uc.420-具有抗肝纤维化作用，lnc uc.420-可能成为肝纤维化治疗的新靶点。通过该研究，培养硕士研究生1名，博士研究生1名。