组蛋白去甲基化酶FBXL11基因对哮喘ASMC增殖和功能的调控作用及分子机制

The unnormal regulation of airway smooth muscle cell proliferation,apoptosis, phenotypic modulation, migration and secretion of airway smooth muscle cell plays a vital role for airway remodeling and development of asthma disease. Recent studies report that FBXL11 functions as a histone demethylation negatively regulated the cell proliferation and activity of NF-κB. Our primary experiments showed us that the FBXL11 mRNA display a trend of down-regulation in asthmatic murine airway smooth muscle, yet few papers were detected on the regulation roles of FBXL11 for proliferation, apoptosis, phenotypic modulation, migration and secretion, and calcium channel of airway smooth muscle. Basing on these reports and our primary results, we predict a hypothesis that FBXL11 plays a critical role in regulating airway smooth muscle cells (ASMCs) remodeling on controling cell proliferation, apoptosis , phenotypic modulation, migration and secretion, and calcium channel of airway smooth muscle through NF-κB pathway. In order to testify this hypothesis, through in vio and in vitro experiments,we will detect the NF-κB activity and cell proliferation, apoptosis, phenotypic modulation, migration and secretion, and calcium channel of airway smooth muscle through over-expression and RNAi of FBXL11 on airway smooth muscle of normal and asthmaic murine model using methods of real-time PCR, western blot, immunohistochemistry, gne expressiong chip, patch-clamping techniques, and other electrophysiological techniques, respectively in cell, protein and gene interaction net levels. The results gaining from this project will give us an insight of understanding the molecular mechanism of function regulation for airway smooth muscle, and also will be helpful for improving symptoms of airway remodeling and new drugs screening for prevention and cure of asthma.

气道平滑肌细胞（ASMC）增殖、凋亡、表型转化、迁移、分泌及钙通道等功能的异常改变是引起哮喘气道重塑的关键因素。研究表明FBXL11负调控NF-κB通路及细胞增殖，但有关FBXL11对哮喘ASMC功能的影响尚少见有报道。申请人在前期工作基础上，开展小鼠哮喘模型在体及离体实验研究，确认组蛋白去甲基化酶基因FBXL11对哮喘ASMC的结构和功能的调控作用；在此基础上，用构建慢病毒载体的方法使FBXL11基因表达沉默或过度表达，采用定量PCR、蛋白印迹、膜片钳、体内外转染及免疫电生理等技术，从细胞、蛋白、基因网络等不同水平研究其对哮喘ASMC功能结构、钙通道蛋白、膜电位等的影响及其信号转导机制；同时，利用构建的shRNA慢病毒载体或过表达载体对FBXL11基因靶向干预，抑制或增加FBXL11表达，达到调控ASMC功能、改善气道重塑的目的，为探寻哮喘发病机制及后续基因干预治疗提供理论依据。

哮喘作为一种难治性呼吸系统疾病，目前还缺乏有效治疗手段和药物。在哮喘发病过程中，气道平滑肌细胞的异常增殖是其主要病理特征之一。因此，探讨哮喘气道平滑肌细胞增殖的分子调控机制对寻找哮喘疾病治疗新的靶标具有十分重要的意义。本项目的前期研究表明：一种编码组蛋白去甲基化酶基因FBXL11基因可能在调控平滑肌细胞增殖中发挥重要功能。因此，阐明FBXL11在哮喘发病过程中表达及对细胞增殖调控作用，并揭示两者之间的机制将对探明FBXL11基因功能及为探寻哮喘疾病新的治疗靶标奠定理论基础。. 本项目首先基于半定量、荧光定量、Western Blot、免疫组化技术从转录水平、蛋白水平、组织水平分析了FBXL11基因在卵清蛋白致敏的急慢性哮喘小鼠气道组织尤其是气道平滑肌组织中的差异表达和组织分布情况，发现了FBXL11基因在急慢性哮喘小鼠气道组织中均呈下调的表达模式，尤其在慢性哮喘小鼠中其表达量最低，该研究提示了FBXL11表达可能与气道组织平滑肌细胞增殖之间存在某种关系。为进一步揭示FBXL11基因与细胞增殖之间调控关系及对气道平滑肌细胞增殖的调控分子网络，我们进一步利用基于RNA-seq转录组测序技术分析了急慢性哮喘气道平滑肌组织间基因差异表达情况，基因网络分析表明除了参与炎症反应的通路以外，差异表达基因均集中于与细胞增殖相关的NF-κB信号通路和PI3K-AKT通路。与此同时，我们通过构建的多个FBXL11突变的293T细胞株开展了FBXL11基因对细胞增殖影响及机制的研究，研究结果发现FBXL11蛋白的缺失不仅导致了细胞整体组蛋白H3K36修饰水平的紊乱，同时也显著抑制了细胞的增殖并促进细胞凋亡。该研究结果提示我们FBXL11可能通过影响NF-κB通路和PI3K-AKT通路及细胞凋亡相关通路在调控哮喘气道平滑肌增殖中起作用。. 本项目对FBXL11的表达与哮喘气道平滑肌增殖及分子调控网络进行了解析，对基于FBXL11为靶点的哮喘疾病的防治药物的开发具有潜在的意义。本项目已发表SCI论文3篇，中文核心期刊论文1篇。