0610009E02Rik/Notch1在HVOD损伤肝细胞修复中的调控作用研究

The hepatic veno-occlusive disease (HVOD) is the severe complication after bone marrow stem cell transplantation. The risk and the degree of HVOD is dependent on the repair of the hepatic cells. Notch1 signaling is one of the key signaling pathways in the reparation of the hepatic cells. Previous studies mainly focused on the regulation of the Notch1 downstream signaling pathway. However, the regulative mechanism of Notch1 itself expression is still unclear. The long non-coding RNAs provide a new view of the gene expression regulation. Our studies indicate that the lncRNA 0610009E02Rik displays sequence similarity to the Notch1. Importantly, both the expression of the 0610009E02Rik and Notch1 is dawnregulated at the same time and display positive correlation in the hepatic cells which are obtained from bone marrow stem cell transplantation caused liver damaged mice models. All these results suggest that the lncRNA 0610009E02Rik is a positive regulator of Notch1 which expression has significantly effect on the reparation of the hepatic cells. In the project, we plan to elucidate the roles of the lncRNA 0610009E02Rik in the reparation of the hepatic cells and the regulative mechanisms of Notch1 gene expression from the DNA methylation, histone modification and protein expression level in the in vivo and in vitro hepatic cells damaged models. If successful, this study will broaden our vision on the acknowledgements of the Notch1 signaling and the repair mechanism of the hepatic cells in the HVOD.

肝静脉闭塞病是造血干细胞移植的严重并发症，其发生风险及严重程度受肝细胞修复程度的影响。Notch1信号是肝细胞修复的重要信号通路之一。既往研究侧重于Notch1通路的下游调控，而其自身表达调控尚待探索。长链非编码RNA（lncRNA）是基因表达调控的新着眼点，我们发现lncRNA 0610009E02Rik与Notch1同源；造血干细胞移植小鼠肝损伤模型中肝细胞内0610009E02Rik与Notch1同步下降，二者呈正相关。提示该lncRNA是Notch1的正性调节子，可调控Notch1表达影响肝损伤的修复。本课题拟采用肝细胞损伤离体和在体模型，明确0610009E02Rik在损伤肝细胞修复中的作用，从DNA甲基化、组蛋白修饰及蛋白翻译等方面探索其调控Notch1表达机制，拓宽人们对Notch1信号通路的认识，深入解析肝静脉闭塞病中肝修复的机理，为临床多种肝损伤相关疾病的治疗提供新视角。

肝静脉闭塞病是造血干细胞移植的严重并发症，其发生风险及严重程度受肝细胞修复程度的影响，深入探讨肝细胞损伤修复机制尤为重要。前期研究发现Notch1参与该损伤修复过程，但具体机制尚不明确。 本课题主要研究肝细胞内长链非编码 RNA 0610009E02Rik 对 Notch1 受体表达的调控机理，进一步明确 0610009E02Rik 对HSCT- HVOD 中损伤肝细胞修复的调控作用。利用成功构建的长链非编码RNA 0610009E02Rik竞争抑制和过表达载体，研究发现，Rik影响正常肝脏的生物学功能，参与损伤肝细胞的增殖，而且通过调控Notch1参与损伤肝细胞的增殖修复。输注Rik竞争抑制和过表达载体，建立同基因/异基因造血干细胞移植模型，在上调或下调肝细胞中lncRNA0610009E02Rik的的情况下，分析肝功能、肝脏病理以及肝指数，发现lncRNA0610009E02Rik可促进移植后肝脏损伤修复，促进肝细胞增殖相关因子PCNA, Ki67和HGF的表达。进一步体内外实验发现，lncRNA0610009E02Rik可明显增加肝细胞中Notch1信号通路及其下游多种增殖分化相关靶基因的表达，使得移植早期阶段肝细胞增殖作用明显，利于损伤肝细胞的修复。二者呈正相关性。通过核浆分离实验可确定lncRNA 0610009E02Rik在细胞核发挥作用，提示lncRNA 0610009E02Rik可能在转录水平调控Notch1的表达。荧光素酶实验和micro RNAarray 筛查实验发现二者不存直接相互作用关系且与microRNA无关。RNA pull down实验证实lncRNA 0610009E02Rik可能通过与ZNF-385A蛋白相互作用来调控Notch1受体的表达。但具体机制有待深入探索。本研究拓宽人们对 Notch1 信号通路的认识，深入解析肝静脉闭塞病中肝修复的机理，为临床多种肝损伤相关疾病的治疗提供新视角。