长链非编码RNA–p21通过Notch1通路抑制百草枯中毒后的肺上皮-间质转化机制研究

Pulmonary fibrosis is the key dead reason of paraquat (PQ) poisoning, but there is no specific antidote. Investigation by our group showed that Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms of pulmonary fibrosis after PQ poisoning. And Notch1 signaling plays an important role in EMT induced by PQ. But until now, there is no study on upstream regulation factors of Notch1 signaling. Studies showed that long chain non-coding RNA p21 (lincRNA-p21) was related to tissue fibrosis. So, in this study, we will explore the expression changes of lincRNA-p21 after EMT induced by PQ, and the changes of Notch1 and EMT after silence or overexpression of lincRNA-p21. Then we will build transgene mice that overexpress lincRNA-p21, and detect the expression of Notch1, EMT and fibrosis in mice. Meanwhile, we will detect miR-6875-3p, which is the most possible upstream factor of lincRNA-p21. As our knowledge, this is the first investigation on function of lincRNA-p21 in pulmonary fibrosis caused by PQ. It will provide new direction for PQ poisoning mechanism, and may supply new treatment target.

百草枯中毒后发生的迟发型肺间质纤维化是患者死亡的重要原因，目前尚无有效解毒剂。课题组既往研究显示上皮-间质转化（EMT）是百草枯致肺间质纤维化的重要原理之一，Notch1通路在百草枯致EMT中发挥了重要作用。但Notch1通路发挥作用的上游调控因子尚不清楚。研究显示长链非编码RNA p21(lincRNA-p21)与组织纤维化相关。因此，本项目研究百草枯致肺上皮细胞EMT后lincRNA-p21的表达变化，沉默或过表达细胞lincRNA-p21后对Notch1表达及EMT的影响。同时，我们将探索调控lincRNA-p21的上游因子miR-6875-3p。课题组将进一步采用转基因技术构建lincRNA-p21过表达的小鼠模型，研究百草枯中毒后模型小鼠肺内EMT及纤维化是否有所改善。这是目前为止首例对lincRNA-p21在百草枯致肺间质纤维化中作用的研究，为百草枯研究提供新的方向。

百草枯（Paraquat, PQ）中毒的死亡率居高不下，PQ可在多种器官内分布，但主要具有明显的肺毒性，其引起的不可逆的肺纤维化是PQ中毒的主要死因。上皮-间质的转化（epithelial-mesenchymal transition，EMT）是指在一定的病理生理条件下上皮细胞表型发生改变，转化为间质细胞的过程。在此过程中，细胞上皮标志物如E-cadherin，而表达间质的标记物如α-SMA等表达上升。EMT可参与肺间质纤维化的过程。研究中，我们通过全转录组分析百草枯中毒后长链非编码RNA、microRNA、mRNA的表达情况。预测了导致肺上皮细胞发生EMT的CeRNA调控网络。有差异性表达的microRNA 1076个，差异性表达的lncRNA 558个。其中我们验证了HOTAIR-miR-17-5p-MMP2的CeRNA调控网络对EMT作用。HOTAIR可以作为分子海绵吸附miR-17-5p，从而使miR-17-5p对MMP2的抑制作用减弱，促进肺上皮细胞EMT的发生。mmu-miR-23a-5p/IL22BP促进百草枯所致的肺上皮细胞EMT。研究结果显示，IL22BP在PQ所致的肺上皮细胞EMT中起保护作用，PQ通过IL18/1L22BP致小鼠肺上皮发生间质转化，而IL22BP 是miR-23a-5p的靶基因，miR-23a-5p/1L22BP调控机制参与了百草枯中毒后肺脏上皮细胞EMT过程。对lncRNANONMMUT100623.1/miRNA 9-8974-5p/ RGS2调控网络研究显示miRNA 9-8974-5p抑制RGS2mRNA表达，促进EMT发生。敲除lncRNANONMMUT100623.1后RGS2表达下降，EMT过程增强。上述研究为百草枯中毒肺纤维化的发生提供理论依据，为百草枯所致肺肺纤维化的救治提供可能的靶点。