lncRNA 2700086A05Rik吸附miR-1236介导百草枯致肺纤维化过程的新机制研究

lncRNA A05Rik was a novel long non-coding RNA identified from our previous subject. Which can up-regulated Paraquat induced pulmonary fibrosis process through mediated target gene HOXA3. However, HOXA3 wasn’t the exclusively pathway for A05Rik’s regulation. Combining the clue from Pull down results, the AGO-RIP as well as the RNA-Seq, we obtained a miRNA, miRNA-1236, which had several binding site with A05Rik, was significant lower expressed in Paraquat treated animal model and cells line samples. Preliminary data showed that miRNA-1236 target gene IGFBP5 was a key factor for the EMT regulation. Silence of A05Rik led to the higher expression of miR-1236, and lower expression of IGFBP5, thus reversed the EMT process, and that was happened without HOXA3. Those finding suggest that A05Rik/miR-1236/IGFBP5 pathway may accelerate Paraquat induced pulmonary fibrosis. The current study aims to illuminate the mechanism of adsorption effect between A05Rik and miR-1236, and the following IGFBP5 regulation both in vivo and in vitro using cross gain and loss-of-function approaches and might provide novel and integrated mechanism on ncRNA-ncRNA network in Paraquat induced pulmonary fibrosis and therapeutic strategies for treatment of the disease on its early stage.

lncRNA A05Rik是前期课题筛选获得，能通过调控靶基因HOXA3促百草枯致肺纤维化发展的一条新长链非编码RNA，但HOXA3非其唯一调控途径。通过Pulldown实验线索、AGO-RIP技术以及测序结果，我们筛选获得与A05Rik存在结合位点、低表达于百草枯动物和细胞模型的miRNA-1236。初步研究发现调控EMT关键基因IGFBP5是miRNA-1236下游靶基因；沉默A05Rik可致miR-1236表达升高、IGFBP5表达下降同时逆转EMT过程，且不受HOXA3影响。故提出A05Rik/miR-1236/IGFBP5通路促百草枯致肺纤维化的科学假说。本项目拟结合体内体外，交叉过表达/沉默策略，阐明lncRNA A05Rik吸附miR-1236调控IGFBP5表达的分子机制。有助于完整了解ncRNA-ncRNA网络参与百草枯致肺纤维化的机制，为治疗此疾病提供新靶标的证据。

百草枯是一种世界范围内广泛使用的高效能除草剂，同时也是危害国人健康，造成急性中毒，且死亡率最高的毒物之一。肺脏是百草枯最易受累的主要器官，百草枯产生的急性炎症损伤及快速纤维化异常修复所造成的呼吸衰竭是其致死的最主要病因。本研究旨在观察lncRNA A05Rik参与百草枯致肺纤维化的过程及机制。RT-PCR证实A05Rik在动物纤维化组织以及TGFβ1或百草枯诱导的HBEc及A549的EMT细胞模型中均高表达，并且在此过程中发生明显胞浆聚集。说明A05Rik分布在胞浆内，参与了百草枯等多种致纤维化过程的调控。A05Rik 通过siRNA沉默后，可以抑制TGFβ1和百草枯诱导HBE及A549发生EMT过程；与空载对照相比，过表达A05Rik转染HBE或A549细胞，可使上皮细胞标记物E-cadherin明显下降；而间充质细胞标记物vimentin等明显升高；Co-IP实验证实了A05Rik与HOXA3确有结合。WB示过表达A05Rik转染HBE或A549细胞后，均可以抑制HOXA3的表达；观察发现沉默HOXA3后，HBEc及A549细胞均可自发性出现EMT趋势；同时用siRNA HOXA3和siRNA A05Rik干扰细胞后，HBEc及A549细胞致EMT的效率明显下降；通过携带目的序列A05Rik siRNA或对照乱序的腺相关病毒AAV9经气道给药，可以在28天观察时间内有效转染肺脏并引起肺内A05Rik 的表达抑制，同时引起了靶基因Hoxa3表达升高，从生存率、体重趋势、肺泡支气管灌洗液细胞分类、肺功能静态顺应性及弹性阻力、BALF和血清的TGFβ1水平、羟脯氨酸浓度等指标，以及组织病理学观察各组纤维化程度、组织免疫荧光观察EMT标记物表达差异观察：A05Rik表达抑制改善了百草枯致肺纤维化小鼠的表型。lncRNA A05Rik通过靶向调控基因HOXA3，促进上皮-间充质细胞转分化过程，从而促进肺纤维化的病理进程，通过肺组织内A05Rik 表达抑制可以显著改善百草枯致肺纤维化小鼠的表型。lncRNA A05Rik在百草枯致肺纤维化过程中起关键作用。