LncRNA通过调控TGF-β1/Smad3信号通路在矽肺纤维化中的分子机制研究

Silicosis is a severe occupational disease that poses great threats to professional health. The character of it is diffuse pulmonary interstitial fibrosis, the mechanism of it has not been fully understood. many studies indicate it’s the reaction of myofibroblast proliferation caused by the interaction of cytokine. TGF-β1/Smad3 signaling pathway is recognized fibrosis way of silicosis. LncRNAs play an important role in both transcriptional and post-transcriptional regulation of genes. Whether or not regulate key molecular signals on the TGF-β1/Smad3 pathway involved in silicosis fibrosis need to be studied. Early research of this study showed differential expression profiles of lncRNAs between silicosis and normal lung tissue. Base on this, we hypothesized lncRNAs are able to intervene silicosis fibrosis. Bioinformatics software was applied to predict lncRNAs involed in the regulation of key molecular on TGF-β1/Smad3 signal pathway. Hypo-expression and hyper-expression models in vitro of two lncRNAs will be established to observe the impact of lncRNAs on key molecular on the signal pathway from gene and protein level. Which would verify lncRNAs participate in TGF-β1/Smad3 signal pathway and thus regulate the process of fibrosis of silicosis, probably shed some light on the association of lncRNAs with silicosis and its therapy from a new perspective as well。

矽肺是严重危害劳动者健康的职业病，患病特征是弥漫性肺间质纤维化，发生机制尚未阐明。多认为是细胞因子相互作用引起的成纤维细胞增殖反应，TGF-β1/Smad3信号通路是公认的矽肺纤维化通路。lncRNA能够调控转录和翻译过程，是否能够调控TGF-β1/Smad3信号通路参与矽肺纤维化过程有待研究。前期研究发现矽肺组织和正常肺组织LncRNA表达谱差异，据此假设lncRNA能够干预矽肺纤维化过程。本课题拟采用生物信息学软件预测可能作用于TGF-β1/Smad3信号通路关键分子的lncRNA，建立lncRNA的过表达和低表达细胞模型，从基因和蛋白水平观察lncRNA对TGF-β1/Smad3信号通路关键分子的影响，证明lncRNA能够调控TGF-β1/Smad3信号通路参与矽肺纤维化过程，从新的视角阐明lncRNA与矽肺纤维化关系，并为矽肺的防治提供新的思路。

矽肺是严重危害人类健康的职业病，位于法定职业病之首。是由于长期吸入含有游离二氧化硅(SiO2)粉尘而导致的以肺组织广泛性纤维化为主要特征的全身性疾病。矽肺发病过程极其复杂，致病因素明确是矽尘颗粒，最终结果是肺组织逐渐被细胞外基质替代引起肺实变而使患者丧失呼吸功能。已知TGF-β1/Smad3信号通路在矽肺纤维化中起关键作用。本研究期望从长链非编码 RNA(long non-coding RNA，lncRNA)能够调控基因转录及翻译的角度，寻找能够干预TGF-β1/Smad3信号通路上关键分子的lncRNA，进而干预矽肺纤维化的进程。通过非暴露气管一次性注入法复制矽肺大鼠模型，肺组织HE染色和Masson染色，免疫组织化学染色鉴定矽肺模型复制成功。选择TGF-β1/Smad3抑制剂SB431542干预信号通路，体内、体外验证TGF-β1/Smad3信号通路在矽肺纤维化过程中的作用基础上。结合矽肺特异mRNA表达谱芯片选定TGF-β1/Smad3信号通路上关键分子Tgfbr1，生物信息学技术分析可能对Tgfbr1有靶向调控作用的miRNA，利用miRNA和lncRNA在体内的竞争机制反向预测可能作用于miRNA的lncRNA。对生物信息学预测发现的21个lncRNA逐一验证，根据它们与Tgfbr1表达的相关性缩小选定的lncRNA范围，FISH实验观察各lncRNA的亚细胞定位，选定在细胞质中表达的lncRNA。然后通过粉尘刺激肺泡巨噬细胞，并与肺泡上皮细胞共培养（体外矽肺模型），体外干扰lncRNA后，观察下游EMT关键分子和纤维化因子的改变，探讨lncRNA是否通过调控Tgfbr1进而影响矽肺纤维化过程。研究结果发现了矽肺纤维化过程中特有的lncRNA和mRNA表达谱，慢病毒干扰肺泡上皮细胞中lncRNA MRAK050699的表达后，上皮细胞间质转化相关因子Snail家族，ZEB家族，间质细胞相关蛋白a-SMA、Vimentin、N-cadherin，纤维化因子COL1、COL3和FN的基因和蛋白表达水平均发生明显的变化。证明lncRNA MRAK050699能够调控TGF-β1/Smad3信号通路上关键分子Tgfbr1参与矽肺纤维化过程，从新的视角阐明lncRNA与矽肺纤维化关系，并为矽肺的防治提供新的思路。