p66Shc在肝纤维化发病中的作用及lncRNA-Mical2/miR-203a-3p对其调控研究

Oxidative stress is critical to liver fibrosis. Our studies have demonstrated that the p66Shc-mediated oxidative stress participates in the hepatic epithelial-to-mesenchymal transition，and the liver fibrosis in animals and humans are positively correlated with the p66Shc expression. We further found that 1) Depletion of p66Shc by RNA interference significantly inhibits reactive oxygen species (ROS) generation and hepatic stellate cell activation; 2) p66Shc can be inhibited by miR-203a-3p, which is significantly reduced in liver fibrosis; Bioinformatics further predicate that lncRNA-Mical2 has the ceRNA interaction with miR-203a-3p, and is negatively correlated with miR-203a-3p expression. Accordingly, we put forward the hypothesis that p66Shc plays a vital role in liver fibrosis, and inhibition of p66Shc by lncRNA-Mical2/ miR-203a-3p protects against liver fibrosis. In this study, by utilizing gene model animal and RNA intervention, we investigate the significant role of lncRNA-Mical2/miR-203a-3p-p66Shc pathway in liver fibrosis and intend to provide a novel pharmacological target for the treatment of liver fibrosis.

氧化应激是肝纤维化损伤的关键环节。我们研究首次发现：p66Shc介导氧化应激参与肝上皮间质转化，动物及人肝纤维化病变与p66Shc表达正相关。进一步发现：1）干扰p66Shc可显著抑制细胞内ROS产生及肝星状细胞激活，抑制纤维化；2）miR-203a-3p可靶向抑制p66Shc，且在肝纤维化表达显著降低；信息学预测lncRNA-Mical2和miR-203a-3p具有ceRNA关系，其与miR-203a-3p在纤维化表达负相关。据此提出科学假说，p66Shc在肝纤维化中发挥重要作用，通过lncRNA-Mical2与miR-203a-3p的ceRNA网络抑制p66Shc的表达及活性，可有效防治肝纤维化。本研究利用基因敲减及过表达体内外模型，采用多种分子生物学技术阐明p66Shc及lncRNA-Mical2/miR-203a-3p调控在肝纤维化发病中的机制，为肝纤维化防治及药物研发提供新靶标。

氧化应激是促进肝纤维化发生发展的重要机制。非编码RNA (ncRNA)在调节肝纤维化中发挥重要作用。ceRNA网络是ncRNA介导基因调控的主要机制。本研究利用四氯化碳小鼠肝纤维化动物模型以及原代肝星状细胞、LX2等细胞模型，采用荧光素酶报告基因、qRT-PCR、RNAi等分子生物学技术，首次阐明衔接蛋白p66Shc介导的线粒体氧化应激在肝纤维化发病中的重要调控作用及其lncRNA-Mical2/miR-203a-3p的ceRNA网络调控机制。. 我们的研究发现：1）p66Shc作为线粒体ROS关键的调节因子，在肝纤维化中表达显著增加，动物及人肝纤维化程度与p66shc表达正相关；特异性抑制p66Shc的表达，可显著减少肝星状细胞（HSC）ROS的产生，抑制HSC的激活、增殖和上皮间质转化，降低胶原沉积，减轻肝纤维化。2）p66Shc通过调节线粒体ROS，激活NLRP3炎性体，促进肝星状细胞激活；酚酸类化合物鼠尾草酸可下调p66Shc，抑制ROS产生，促进β-catenin磷酸化，抑制肝星状细胞增殖，减轻肝纤维化。3）miR-203a-3p通过靶向p66Shc，抑制肝星状细胞激活及增殖，减轻肝纤维化。 lncRNA Mical2作为ceRNA，抑制miR-203a-3p对p66Shc的靶向调控，上调p66Shc 的表达，促进肝纤维化的发生。. 本项目发现p66Shc介导的线粒体氧化应激在肝纤维化发病中的重要调控作用，并通过体内外实验，应用分子生物学、生物信息学等技术，阐明lncRNA Mical2/miR-203a-3p的ceRNA网络及酚酸类化合物对其的调控作用。本研究有助于完善肝纤维化中p66Shc的调节机制，针对p66Shc的靶向调控，可为肝纤维化疾病提供防治策略和治疗新靶标。