lncRNA调控TGF-β1在纳米NiO致肺纤维化中的作用及分子机制

Nano NiO, as a new functional nanomaterials, led to pulmonary fibrosis in animal model. Our previous study found that the potential mechanisms of pulmonary fibrosis induced by nano NiO related to the activatd TGF-β1, occurrence of epithelial-mesenchymal transition (EMT) and up-regulated lncRNA. Studies confirmed that lncRNA played a key role in pulmonary fibrosis. However, the study have been performed unclearly in order to understanding the regulating mechanisms of lncRNA, TGF-β1, EMT and PI3K/AKT pathway in pulmonary fibrosis caused by nano-NiO. Based on the established model of A549 cytotoxicity induced by nano-NiO, this project will identify the lncRNAs which regulate TGF-β1, and further elucidate the mechanism of lncRNA regulating TGF-β1 which mediate EMT and PI3K/AKT signaling pathway using techniques of lncRNA lentiviral plasmid transfection, siRNA interference, TGF-β1 and PI3K blockers，quantitative PCR, western blot etc., and validated the above results by animal experiments and population study. The project can provide a scientific basis for elucidating the molecular mechanisms of pulmonary fibrosis induced by nano-NiO, and finding the early biomarkers of lung injury induced by nano-NiO and safety evaluation.

纳米氧化镍（NiO）作为新型功能纳米材料可致实验动物肺纤维化，我们前期研究发现大鼠肺纤维化可能与肺组织TGF-β1表达上调、上皮细胞间质化（EMT）发生及lncRNA表达改变有关。研究证实lncRNA与肺纤维化发生发展密切相关，但有关lncRNA、TGF-β1、EMT和PI3K/AKT通路在纳米NiO致肺纤维化中的作用及调控机制尚不清楚。本项目在已建立的纳米NiO所致A549细胞毒性模型的基础上，明确调控TGF-β1的lncRNA，采用lncRNA慢病毒质粒转染、siRNA干扰、TGF-β1和PI3K抑制剂、定量PCR和免疫印迹等技术，阐明lncRNA调控TGF-β1介导EMT和PI3K/AKT信号通路在纳米NiO致肺纤维化中的作用及其调控机制，并通过整体动物实验和人群研究进行验证。该项目为阐明纳米NiO致肺纤维化的分子机制、探寻其致肺损伤的早期生物标志及安全性评价提供科学依据。

纳米NiO可致肺纤维化，但有关 lncRNA、TGF-β1、EMT和信号通路等在纳米NiO致肺纤维化中的作用机制尚不清楚。本研究在建立纳米NiO致大鼠肺纤维化模型和细胞胶原沉积模型基础上，发现TGF-β1抑制剂SB431542可抑制纳米NiO引起的A549细胞PI3K/AKT通路活化、EMT和胶原沉积，同时PI3K抑制剂LY294002可降低纳米NiO上调的胶原蛋白水平；过表达的lncRNA MEG3可抑制细胞TGF-β1表达、EMT、PI3K/AKT通路活化及胶原沉积。结果表明，MEG3通过抑制TGF-β1介导的PI3K/AKT通路和EMT来减轻纳米NiO诱导的大鼠肺纤维化。p38通路抑制剂SB203580可逆转纳米NiO所致的A549细胞炎性因子的异常表达，同时过表达的MEG3可抑制纳米NiO诱导的p38通路活化和炎性因子的分泌，表明纳米氧化镍通过下调肺组织lncRNA MEG3表达而激活p38通路而致大鼠肺组织炎性损伤。Hh通路抑制剂CDG-0449可促进A549细胞自噬过程，而雷帕霉素则减少A549细胞胶原沉积；过表达的MEG3可抑制细胞Hh通路激活、增强自噬活性及减少胶原形成，说明MEG3通过调控Hh通路介导的自噬来抑制纳米NiO诱导的肺纤维化。DNMT 抑制剂5-AZDC可减弱纳米NiO诱导的BEAS-2B 细胞PRKCB2表达、JNK/c-Jun通路的激活和胶原沉积，而JNK/c-Jun通路抑制剂SP600125可减轻纳米NiO诱导的细胞胶原沉积；过表达的lncRNA HOTAIRM1可抑制纳米NiO诱导的细胞PRKCB的低甲基化、JNK/c-Jun通路激活和胶原沉积，表明HOTAIRM1可通过抑制RKCB DNA甲基化介导的JNK/c-Jun通路来阻抑纳米NiO导致的肺纤维化。该研究成果可为纳米NiO安全性评价及肺纤维化的早发现、诊断和防控提供理论依据。