LncRNA-GM16685通过cis调控IL-7激活HSC促进日本血吸虫病肝纤维化的分子机制

Hepatic fibrosis is one of the emphases and difficulties of current schistosomiasis control. LncRNA is involved in the regulation of key genes and pathways in other chronic liver diseases, but it has not been reported whether it plays a role in the hepatic fibrosis of schistosomiasis. It is suggested that LncRNA-GM16685 will promote hepatic fibrosis of schistosomiasis by cis regulating IL-7 and then activating HSC according to the preliminary experiments. Therefore, the subcellular localization of LncRNA-GM16685 in liver tissue of mice hepatic fibrosis of schistosomiasis will be detected by in situ hybridization. RNA-pull down experiment and RIP experiment will be performed to screen the LncRNA-GM16685 binding protein, followed by identification of the region using EMSA. Then it will be identified whether the LncRNA-GM16685 fragment-protein complex acts on the IL-7 promoter using luciferase system. In addition, the effect on the activation of HSC and the IL-7 gene expression level of LncRNA-GM16685 in mice will be observed in vitro. Finally, we will observe its role in liver fibrosis of schistosomiasis using Schistosoma japonicum infected mice model. Through the above studies, the role and mechanism of LncRNA-GM16685 in hepatic fibrosis of schistosomiasis will be clarified, and it will provide new clues for the prevention and treatment of schistosomiasis hepatic fibrosis.

血吸虫病肝纤维化是我国当前血防工作的重点和难点之一。研究证实LncRNA参与调控其它慢性肝病，但在血吸虫病肝纤维化中的调控作用尚不明确。本项目前期预实验结果提示LncRNA-GM16685可能通过cis调控IL-7激活HSC从而促进日本血吸虫病肝纤维化。为此本研究将先证实LncRNA-GM16685在血吸虫病肝纤维化组织中的亚细胞定位，然后利用RNA-pulldown实验和RIP实验筛选出其结合蛋白，通过EMSA鉴定其与蛋白的结合区域，并通过荧光素酶系统验证LncRNA-GM16685片段-蛋白复合体是否作用于IL-7启动子，随后体外实验研究LncRNA-GM16685对IL-7的cis调控能力，最后通过体内实验研究LncRNA-GM16685对小鼠血吸虫病肝纤维化的调控作用。本研究旨在阐明LncRNA-GM16685在血吸虫病肝纤维化的作用及其机制，为血吸虫病肝纤维化的防治提供新的线索。

血吸虫病肝纤维化是我国当前血防工作的重点和难点之一。研究证实LncRNA参与调控其它慢性肝病，但在血吸虫病肝纤维化中的调控作用尚不明确。本研究通过RNA-Seq及定量筛选出LncRNA-GM16685作为目标LncRNA分子，以进一步研究其功能及机制。利用原位杂交实验、核浆分离实验及定量PCR实验鉴定出GM16685主要定位在KCs的胞浆，利用RNA-pull down实验和RIP实验鉴定出GM16685与ASF可相互作用，利用EMSA实验鉴定出GM16685的第三个外显子区与ASF蛋白相互结合。体外干扰GM16685，明确其可结果显示经干扰后，HSC中α-SMA和colⅠ基因的mRNA和蛋白表达水平显著下调，ASF基因表达水平和蛋白表达水平均下调，而IL-7的表达水平无明显变化。制备日本血吸虫感染小鼠模型，于感染后第六周通过尾静脉注射Ad-shGM16685注入小鼠模型体内，结果表明GM16685干扰后对感染小鼠的虫荷、卵荷、肝脏重量没有明显影响，但小鼠肝脏大体外观明显改善，肝脏表面虫卵结节减少，HE染色结果显示shGM16685组小鼠肝脏虫卵肉芽肿中浸润的炎性细胞明显减少，肝细胞坏死明显减轻，与感染对照组相比，shGM16685组小鼠肝脏虫卵肉芽肿面积明显减小,纤维化面积也减少shGM16685组小鼠血清中转氨酶AST和ALT水平均明显低于感染对照组。而且shGM16685处理后与感染对照组相比小鼠肝脏组织中LncRNA-GM16685和ASF的mRNA水平明显下调，而抑炎性细胞因子明显上调。通过以上研究阐明了GM16685-ASF通过促进HSC活化细胞极化从而促进血吸虫病肝纤维化。该研究为血吸虫病肝纤维化的发生发展机制的进一步明确提供新的见解，为研究血吸虫病肝纤维化防治提供了新的思路，并可能为血吸虫病肝纤维化诊断和治疗提供新靶点。