长链非编码RNA H19在肝纤维化中的作用及其分子机制研究

Aberrant activation of hepatic stellate cells (HSC) is key to hepatic fibrosis as well as anti-fibrogenic research. Subsequently to miRNA, long noncoding RNA (lncRNA) is becoming a new hotspot in biomedical research. However, there has been no report about the relationship between lncRNA and hepatic fibrosis yet. H19, which is related to many pathogeneses, is one of the early discovered lncRNA. In previous studies, the applicant found that H19 could be up-regulated by URG11, which was related to chronic hepatopathy; overexpression of H19 significantly enhanced the proliferation of hepatic stellate cell line LX2, while transcriptional inhibition of H19 markedly suppressed the activation of LX2 cells. Further studies revealed that the repression of H19 down-regulated the activity of Smad2 and Smad3, which were key molecules in TGF-β/Smad signaling pathway. Therefore, a hypothesis is proposed: H19 could enhance the signal transduction of TGF-β/Smad pathway, activate HSC and thus promote hepatic fibrosis. To prove this hypothesis, in this study, we will verify the role of H19 in the activation of LX2 cells and TGF-β/Smad pathway, investigate its target protein and sequence structure, identify its pro-fibrogenic role in patients' tissue samples and explore the anti-fibrogenic role of shRNA H19 in rat model. It is prospective that this study will shed light to the understanding of the mechanism of hepatic fibrosis and provide a brand-new approach for the development of anti-fibrogenic drugs.

肝星状细胞（HSC）异常活化是肝纤维化的中心环节和抗纤维化治疗研究的关键靶标。长链非编码RNA（lncRNA）是继miRNA后生物医学领域又一热点，迄今尚无lncRNA与肝纤维化的研究报道。H19是较早发现的lncRNA之一，与多种疾病发生相关。申请人近期研究发现，H19的表达可被与慢性肝病密切相关的URG11上调；过表达H19显著促进肝星状细胞LX2的增殖，抑制H19则显著抑制其活化；进一步研究发现，抑制H19可下调TGF-β/Smad信号通路关键蛋白Smad2、Smad3活性。故推测H19可通过激活TGF-β/Smad通路，活化HSC，进而促进肝纤维化的进展。因此，本研究拟进一步明确H19的促纤维化作用，阐明其相关信号通路，确定H19靶蛋白和调控机制，并在体内观察以H19为靶点的抗纤维化效应。预期成果将为明确肝纤维化发生发展机制提供线索，并为抗纤维化药物研发提供新的方向。

肝纤维化是由急/慢性肝损伤导致的一种可逆性病理改变，表现为细胞外基质在肝组织间质的过度沉积，长期积累将发展为不可逆的肝硬化甚至肝癌，是慢性肝病发展恶化的必经阶段。肝纤维化的控制及逆转在肝病的治疗和预后中起着十分重要的作用。我们在前期发现长链非编码RNA H19与肝纤维化相关的基础上，在人肝星状细胞LX2中进一步明确了H19对肝纤维化标志——星状细胞活化的促进作用，过表达H19可上调α-SMA、Collagen-I以及TGF-β信号通路关键分子SMAD2和SMAD3的磷酸化水平。分子机制层面，发现H19可结合抑制microRNA-148a（miR-148a），从而间接升高miR-148a下游靶标USP4的表达水平，而已知去泛素化酶USP4可降低TGF-β受体泛素化，促进SMAD2的磷酸化。反之，过表达H19的同时转染microRNA-148a的类似物，可以降低H19的促纤维化作用，从而确定了H19 - miR-148a - USP4这一促纤维化调控轴。最后，通过包被H19的慢病毒尾静脉注射入肝纤维化造模小鼠体内，发现小鼠肝脏H19的表达上升可进一步提高肝纤维化水平，从而在体内验证了H19的促纤维化效应。本项目在体内外明确了H19的促纤维化作用并阐明了其具体分子机制。H19有望成为新的靶标，为逆转肝纤维化治疗提供新的方向。