MiRNA在肺损伤与修复中的作用机制研究

【作者】 谢婷；

【导师】 王庆国；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2011， 博士

【摘要】 肺损伤的分子机制研究还十分不完善,尤其在肺纤维化和免疫复合物致肺损伤领域,大量研究工作有待进行。微小RNA (miRNA)平均由22个核苷酸组成,为单链,非编码的RNA。目前认为miRNA是一类非常重要的机体调控因子,成熟的miRNA可以标靶mRNA的3’端非编译区或者编译区,从而沉默标靶基因,并且在诸多病理生理过程中发挥着非常重要的生物调节作用。为了研究miRNA在肺纤维化中的调节作用,我们提取了博来霉素致肺纤维化小鼠模型各个时间点的肺总RNA,采用含1810个探针的miRNA芯片进行miRNA高通量检测,比较博来霉素作用不同时间点与对照组肺总RNA中差异表达的miRNA。研究得到161个与正常组有差异表达的miRNA。同时分析了肺纤维化模型早期和晚期病理进程中差异表达的miRNA。应用生物信息学方法,对不同病理进程中差异表达的miRNA进行了靶点预测分析。这些重要的miRNA包括let-7f, let-7g, miR-127, miR-196b,miR-16, miR-195, miR-25, miR-144, miR-351, miR-153, miR-468, miR-449b,miR-361, miR-700, miR-704, miR-717, miR-10a, miR-211, miR-34a, miR-367,and miR-21.将预测miRNA靶点进行归类分析,得到在肺纤维化重要病理环节,包括凋亡、炎症反应及纤维化中的重要分子通路,如凋亡信号通路、Wnt信号通路、Toll受体和组织生长因子通路中密切相关的miRNA及其靶点基因。我们的研究揭示了肺纤维化过程中不同程度差异表达的miRNA,同时预测出miRNA可能调控的肺纤维化病理过程中重要的分子通路。另外,我们发现miR-127在肺部损伤炎症反应阶段时表达下调。基于此,我们探究了miR-127在肺损伤和炎症中的作用机制。我们发现过表达miR-127的巨噬细胞炎性刺激物作用后,产生的细胞因子[白介素-6(IL-6),白介素1p (IL-1β),组织坏死因子(TNF-α)]释放减少。生物信息学分析发现免疫球蛋白G (IgG) Fcγ受体I(FcyRI/CD64)可能是miR-127的靶点基因。基因芯片,蛋白定量表达分析分别确证了miR-127在mRNA和蛋白水平对CD64的调控。通过构建含CD64-3’UTR的荧光报告质粒,我们明确了miR-127对CD64的直接标靶关系。另外体内实验更进一步发现,鼻内滴注miR-127可以减少IgG免疫复合物(IgG IC)所导致的急性肺内损伤和炎症反应。包括肺组织形态学观察,细胞因子IL-6, TNF-a,巨噬细胞炎性蛋白1β(MIP-1β),巨噬细胞炎性蛋白-2(MIP-2)单核细胞趋化蛋白(MCP-1),趋化因子CXC配体-1(KC)的检测,肺渗透性检测,补体C5a以及急性期应答因子信号传导子及转录激活子3(STAT3)的蛋白水平检测。因此,我们认为miR-127通过标靶巨噬细胞CD64表达,从而减轻肺内IgG IC所致的炎性反应。总之,我们研究了肺纤维化中各特定时间点和特定病理阶段差异表达的miRNA,并且对这些miRNA进行了标靶基因预测,通过将这些标靶基因与特定病理阶段的分子信号通路对应起来,我们分析得到了各个病理阶段miRNA可能发挥作用的分子信号通路。通过对miR-127在炎症反应中的作用机制研究,我们确定了miR-127的作用靶点基因CD64,并发现了miR-127对IgG IC所致急性肺损伤的保护作用。对miRNAs调节肺内纤维化和炎性反应机制的研究揭示了miRNA在调控感染性及非感染性肺损伤炎性反应的重要作用,为今后肺纤维化及肺损伤炎症反应的新药研究提供有力依据。本课题研究了miRNA在肺损伤和修复中的作用机制。第一部分研究内容：博来霉素致肺纤维化的miRNA复杂性分析揭示多途径分子机制研究,撰写论文已发表在Physiological Genomics(影响因子：3.931)杂志上。第二部分研究内容：miR-127调节肺内炎症反应的机制研究,也已被Journal of Immunology(影响因子：6.0)杂志接收,预计在近半年内发表。本研究内容属国内外研究热点,研究方法属国内外领先水平。中医药是世界医学不可缺少的组成部分,在中华传统文明中占有一席之地,是中华人民世代相传的文化宝藏,至今发挥着治疗众多疾病的重大作用。本研究可为今后中医药研究搭建高水平高质量的研究平台,为研究中医药治疗肺部疾病提供有先进的研究方法和技术。更多还原

【Abstract】 The molecular mechanisms of lung injury are incompletely understood, especially in the fields of lung fibrosis and immune complex induced acute lung injury. MiRNAs are short (about 22 bp in length), single-stranded, and non-coding RNAs that inhibit the production of target proteins or induce degradation of mRNAs by binding target mRNAs at complementary sites in 3’-untranslated regions (3’-UTRs) or coding sequences and thereby suppressing target gene expression. MiRNAs are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiological processes. To gain insight into miRNAs in the regulation of lung fibrosis, total RNA was isolated from mouse lungs harvested at different days after bleomycin treatment, and miRNA microarray with 1810 miRNA probes was performed thereafter. MiRNAs expressed in lungs with bleomycin treatment at different time points were compared to miRNAs expressed in lungs without bleomycin treatment, resulting in 161 miRNAs differentially expressed. Furthermore, miRNA expression patterns regulated in initial and late periods after bleomycin were identified. Target genes were predicted in silico for differentially expressed miRNAs, including let-7f, let-7g, miR-127, miR-196b, miR-16, miR-195, miR-25, miR-144, miR-351, miR-153, miR-468, miR-449b, miR-361, miR-700, miR-704, miR-717, miR-10a, miR-211, miR-34a, miR-367, and miR-21. Target genes were then cross-referenced to the molecular pathways, suggesting that the differentially expressed miRNAs regulate apoptosis, Wnt, Toll-like receptor, and TGF- signaling. In addition, we found that miR-127 appeared down-regulated during lung injury. We set out to investigate the role of miR-127 in lung injury and inflammation. Expression of miR-127 significantly reduced cytokine release by macrophages. Looking into the mechanisms of the regulation of inflammation by miR-127, we found that IgG FeγReceptorⅠ(FcγRI/CD64) was a target of miR-127, as evidenced by reduced CD64 protein expression in macrophages over-expressing miR-127. Furthermore, miR-127 significantly reduced the luciferase activity with a reporter construct containing the native 3’-UTR of CD64. Importantly, we demonstrated that miR-127 attenuated lung inflammation in an IgG immune complex (IgG IC) model in vivo. As accessed by lung histology, cytokine release in bronchoalveolar lavage fluid, including interleukin-6 (IL-6), Tumor necrosis factor-α(TNF-α), macrophage inflammatory protein-1β(MIP-1β), macrophage inflammatory protein-2 (MIP-2), Monocyte chemotactic protein-1 (MCP-1) and chemokine (C-X-C motif) ligand-1 (KC), lung permeability assay, compliment component 5a (C5a) and signal transducer and activator of transcription 3 (STAT3) protein assays. Therefore, these data suggested that miR-127 targets macrophage CD64 leading to reduction of lung inflammation.Altogether, we investigated the miRNA expression patterns in lung tissues of bleomycin model, and found the differentially expressed miRNAs during distinct pathologic phases of lung fibrosis. We also predicted gene regulation by differentially expressed miRNAs during lung fibrosis, and cross-referenced to the molecular pathways in distinct pathologic phases of lung fibrosis. We identified for the first time that miR-127 was involved in acute lung inflammation. In particular, we found that CD64 is a bona fide target of miR-127. MiR-127 administration inhibited the inflammatory response of IgG IC induced acute lung injury. We believe that by understanding how miRNAs regulate lung fibrosis and inflammation would pave the way to develop a novel therapeutic approach to the treatment of infectious or non-infectious lung injury.更多还原