miR-149调控矽尘诱导肺纤维化的初步研究

  本文关键词：miR-149调控矽尘诱导肺纤维化的初步研究，由笔耕文化传播整理发布。

        煤工尘肺（Coal workers’pneumoconiosis，CWP）是在生产过程中长期吸入矽尘、煤矽尘或煤尘所导致的以肺组织不可逆性纤维化为主的疾病，主要包括矽肺、煤矽肺和煤肺，是我国最严重的职业病之一。由于发病机制并不完全清楚，目前尚无有效的治疗方法。大量研究表明，矽尘导致的肺纤维化过程包括肺组织炎性损伤、肺组织结构破坏以及伴有基质细胞聚集的肺组织修复。其病理特征为肺组织成纤维细胞过度增殖，多种胶原蛋白在肺组织内过多沉积。其发生发展涉及到大量炎性因子及致纤维化因子的表达以及相关基因的复杂网络调控。近年来，microRNA（miRNA）作为一种非编码小RNA因其广泛的基因调节作用引起了科学界的关注。有研究表明miRNA在炎症、组织发育与分化、细胞增殖、凋亡及组织修复过程中均发挥了重要作用，这些广泛的生物学作用使其成为调节器官表型的关键分子及多种疾病的治疗靶标。目前有关miRNA与疾病之间关系的研究很多，尤其是肿瘤，如肺癌、肝癌、胃癌、前列腺癌、结直肠癌等，此外也包括心肌梗死、心衰、慢性肾病等。但是关于miRNA在尘肺病中的表达谱及功能学研究报道较少。因此，结合我国尘肺病的发病现状，我们进行了煤工尘肺血清miRNA的测序分析，经过生物信息学分析筛检出煤工尘肺与对照表达差异的miRNA，并运用体内试验及体外实验对煤工尘肺差异表达变化一致且最显著的miRNA优先进行了初步的功能学验证，，探讨这些miRNA在矽肺病发生发展中可能的分子机制，期望能将肺纤维化基因表达调控的网络理解提高到一个新的水平，为找出特异性的血清miRNA作为煤工尘肺辅助诊断的生物标志物奠定理论基础，同时也为进一步研究预防和有效治疗尘肺病提供线索。我们主要开展了两个阶段的研究：（1）煤工尘肺病人血清miRNA标志物筛检：即首先通过流行病学调查选择病例和对照、SOLiD测序法筛选出差异表达的miRNA，利用miRWalk综合数据库对候选miRNA所调节的靶基因及信号通路进行了分析；（2）miR-149在SiO2粉尘所致肺纤维化中的功能学研究：在第一阶段研究的基础上，选择其中一种差异表达明显的miRNA（miR-149）进行了初步的功能学验证。第一部分：煤工尘肺特异的血清miRNA筛检目的：通过对CWP病人及接尘对照血清差异表达miRNA进行分析，发现影响CWP发生发展的关键miRNA，为提高尘肺病的早期诊断率及探索新的治疗方法提供科学依据，也为进一步研究尘肺病发病机制提供线索。方法：（1）研究对象的选择：2010年8月通过调查问卷收集某煤矿井下接尘男性工人的年龄、职业史（工龄、工种、防护措施等）、现病史、既往史、个人史等资料，并由专业职业病体检机构对接尘工人进行职业病体检，拍摄高仟伏胸片及DR胸片，根据《尘肺病诊断标准》GBZ70-2009由职业病诊断医师对研究对象进行尘肺病诊断。筛选出煤工尘肺病人27例（壹期、贰期、叁期病人各9例），对照9例，病例与对照在年龄、接尘年代、接尘工龄、工种、吸烟等方面相匹配；（2）全基因组miRNA测序：采集外周静脉血并分离血清，同组血清等量混合，提取RNA，SOLiD测序仪检测全基因组ncRNA表达，应用SOLiD小RNA分析系统（错配、过滤和比对）确定已知的miRNA、新miRNA及表达水平（拷贝数）；（3）靶基因预测及信号通路分析：将测序结果与miRNA数据库比对，筛选出与对照组相比表达变化在2倍以上的miRNA，进一步确定在所有病例组变化方向一致的miRNA，通过miRWalk综合数据库对筛检出的miRNA靶基因进行预测，并对其调控的信号通路进行分析。结果：全基因组测序法对接尘对照组、壹期、贰期、叁期CWP血清中ncRNA进行筛检后，各组鉴定出与数据库匹配的miRNA比例分别为39.9%、42.2%、36.4%、39.9%。以病例组与对照组比较差异大于2倍为筛选条件，壹期、贰期、叁期CWP筛选出的差异表达miRNA数量分别为32、55和39个，其中在所有病例组变化方向一致的miRNA共有5个，表达上调的有miR-25*、miR-9*；下调2倍以上的有miR-20b、miR-222、miR-149，其中以miR-25*及miR-149的变化较为显著。文献报道及靶基因分析显示，这些miRNA可能调节着与细胞增殖、凋亡、炎症、基因转录、细胞周期及信号转导等相关基因的表达。结论：在病例组与接尘对照组血清中，共筛检出5个变化方向一致的miRNA作为候选分子，其中表达明显上调的为miR-25*、miR-9*，表达明显下调的为miR-20b、miR-222、miR-149，这些miRNA可能调控了肺纤维化过程的关键信号通路。第二部分：miR-149在SiO2粉尘所致肺纤维化中的功能学研究目的：结合前期血清miRNA筛检结果，通过体内试验及体外实验研究miR-149在SiO2诱导的肺纤维化中的作用。方法：（1）体内试验：建立SiO2粉尘诱导的小鼠肺纤维化模型，通过qRT-PCR检测miR-149的表达、免疫组织化学及Western blot等实验技术观察小鼠肺组织内miR-149及炎症因子IL-6的动态表达情况；（2）体外实验：①ELISA法检测研究对象血清中IL-6的表达情况；②应用A549细胞及HBE细胞构建SiO2粉尘处理的细胞模型，通过体外转染miR-149mimics及inhibitor使其失调表达，应用qRT-PCR检测miR-149的表达、应用Western blot及ELISA实验观察细胞表达IL-6的情况。结果：（1）与对照组相比，不同期别CWP病人血清中miR-149皆表达下调，IL-6的表达未发生明显变化（P=0.684），但是随着疾病进展，IL-6表达呈上调趋势；（2）在SiO2粉尘诱导的小鼠肺纤维化模型中，免疫组织化学实验结果显示，25g/L SiO2粉尘处理小鼠后的第1周、第2周、第4周，其肺组织中的炎症反应较为明显。在处理第1周，与对照组相比，生理盐水组及粉尘组有多量的炎症细胞浸润；在第2周，生理盐水组的炎症反应逐渐恢复，在第4周时炎症已基本消失，但是粉尘组的炎症反应一直持续，且较重，到第4周时，肺组织内肺泡壁增厚，肺泡结构塌陷；Real-time PCR及Western blot实验分别证实SiO2粉尘处理后的各时间点miR-149表达都明显下调（P<0.000），而IL-6表达明显上调；（3）在粉尘处理的细胞模型中，SiO2粉尘可明显导致A549及HBE细胞凋亡增加，且A549细胞比HBE细胞更加敏感，用不同浓度SiO2粉尘处理细胞后其表达IL-6的水平明显上调，存在明显剂量效应关系，而A549细胞除了具有剂量效应关系外，随着SiO2粉尘处理时间的延长，IL-6的表达水平也逐渐增加，还呈现出时间效应关系；用50μg/ml SiO2粉尘处理A549细胞24h及150μg/mlSiO2粉尘处理HBE细胞12h后，HBE细胞内miR-149的下调非常明显，粉尘处理几乎完全抑制了miR-149的表达（P<0.000），而在A549细胞内miR-149的变化未发现统计学差异，但是可见到miR-149也呈下调趋势（P=0.234）；A549细胞转染miR-149相似剂后使IL-6的表达下调，而转染抑制剂后上调了IL-6的表达水平。结论：通过体内试验及体外实验我们发现：（1） SiO2粉尘处理可明显导致小鼠肺组织的炎症反应；（2） miR-149的下调及IL-6的上调可能参与了SiO2粉尘处理后的炎症、肺纤维化等相关过程；（3） miR-149可以负调控A549细胞表达IL-6的水平。

    Coal workers’ pneumoconiosis（CWP） is characterized by irreversible lungfibrotic diseases resulting from chronic dust inhalation（silica,silica and coal or coaldust）, which mainly includes the silicosis and coal pneumoconiosis. CWP is one ofthe most serious occupational disease in China with no effective therapies. Currentlythe pathogenesis of silicosis is still unknow. The numerous studies have shown thatthe processes of pulmonary fibrosis include inflammatory injury, tissue desdructionand abnormal tissue repair with matrix deposition. The pathological features showincreasing fibroblast proliferation and collagen synthesis in the lung tissue. And itinvolves in a large number of inflammatory cytokines and fibrosis factor expressionand regulation of related genes in complex networks. Recently, microRNAs（miRNAs） as small noncoding RNA molecules of1822nucleotides （nt） in length,play critical roles in various physiological processes such as inflammation, tissuedevelopment and differentiation, cellular apoptosis, proliferation, and tissue repair,which have emerged in the recent decade as key regulators of organ phenotypes andpotential targets for therapeutic interventions in multiple diseases. But there are fewstudies refer to miRNAs in the research of silicosis. Considering the prevalence forsilicosis in China, the present study was about the miRNA screening in serum ofCWP patients and healthy controls, and further functional studies both in vivo and in vitro were also conducted on the candidate miRNA. Hoping to find out thebiomarkers of diagnosis, progression and treatment of CWP, and further provide cluesto reveal the pathogenesis of CWP/silicosis and the roles for certain miRNAs in it.The dissertation is divided into two parts: analysis of serum genome-widemiRNAs for CWP in a case-control study and preliminary study of the role formiR-149in silica-induced pulmonary fibrosis.Part Ⅰ: Analysis of Serum Genome-wide miRNAs for CWP in a Case-ControlStudy.Objective: The key miRNAs which involved in pathogenesis and development ofCWP will be explored by screening the different expression of miRNAs in serumbetween cases and controls, so as to provide further clues to pathogenesis and newtherapies of pneumoconiosis.Methods:（1） The epidemiological information of the research subjects were obtainedby questionnaire, which included age, exposure history, jobs and other relatedepidemiological items. Health examinations including high KV chest X-ray and DRwere conducted by occupational professional agency. CWP patients were diagnosedaccording to the China national criteria for pneumoconiosis （GBZ70-2009）.One control group and three case groups which were CWP stage Ⅰ, stage Ⅱ and stageⅢ （n=9in each group） were mainly matched by age, exposure year, job, diseasehistory, smoking and so on.（2） Equal volume of sera from cases and controls in eachgroup were pooled for SOLiD sequencing-based miRNA profiling.（3） The keymiRNA target genes were primarily selected according to miRNA profiling resultsthat were blast to the known miRNA databases to identify the differently expressedmiRNAs with2-folds change, in which candidate miRNAs with the accordant changetrends in all of the case groups compared to the controls were identified. Genespotentially targeted by the selected miRNAs were identified by using the miRWalkdatabases available online.Results: Compared to the miRBase （15.0）,39.9%、42.2%、36.4%、39.9%knownprecursor miRNAs were identified in control, stage I, stage II and stage III of CWP, respectively. In these effective reads, the most abundant length was22nt size class.Using2-fold change as a cutoff level,32,55and39deregulated miRNAs wereidentified in each stage cases compared to controls, in which two up regulatedmiRNAs and three down regulated miRNAs with an accordant change in all of thecase groups compared to the paired controls were identified （miR-25*,miR-9*upregulated; miR-20b,miR-222and miR-149down regulated）. Target genes predictionand pathway analysis were then conducted for the candidate miRNAs, suggesting thatthe differentially expressed miRNAs regulate proliferation, apoptosis, inflammation,cell cycles, gene transcription and other biological process. Pathway analysis hasshown that candidate miRNAs regulated the key pathways in process of pulmonaryfibosis.Conclusion: Totally two up regulated and three down regulated miRNAs with anaccordant change direction were identified in all of case groups compared to thepaired control. And the candidate miRNA molecules may be involved in the keyprocesses for pulmonary fibrosis.Part Ⅱ: Preliminary Study on the Role of miR-149in Silica-induced PulmonaryFibrosis.Objective: Basing on the miRNA profile results, to explore the potential roles ofmiR-149in the pathogenesis of silica-induced pulmonary fibrosis.Methods: Studies were conducted both in vivo and in vitro to verify the preliminaryresults and to find new mechanisms of miR-149in regulating silica-inducedpulmonary fibrosis.（1） In vivo studies: The expression of miR-149was tested byReal-time PCR and inflammatory cytokine IL-6by immunohistochemistry andWestern blot assay in the lung tissues of silica-induced pulmonary fibrosis mice,.（2）In vitro studies:①The m odels ofSiO2exposed cells （A549and HBE） wereestablished. The A549cells were transfected with miR-149mimics and inhibitor, andthen the expression of IL-6in cells was tested by Western blot assay;②T heexpression of IL-6in CWP patients and paired controls was determined byEnzyme-linked immunosorbent assay. Results:（1） The serum expressions of miR-149in each stage of CWP patients weredown regulated compared to that of controls, but no significant differance was foundin expression of IL-6. However, it was found that the expression of IL-6was upregulated with the progress of CWP.（2） In the mice model, the pulmonary alveoli andinterstitial were infiltrated with amount of inflammatory cells after treated in week1in both silica-treated and saline-treated groups. The inflammation alleviatedgradually in saline-treated mice in2weeks, and was similar to that of controls. Butfor the silica-treated mice, the pulmonary alveolar wall were destoried and interstitialthickened obviously gradually. The expression of miR-149in lung tissues downregulated significantly in any check points（P<0.000）, while IL-6expression was upregulated obviously.（3） In the silica-treated cell models, the apoptosis of A549andHBE cells increased after silica-treated, especially for A549cells which was moresensitive to the silica challenge. For the A549cells, the expression of IL-6increasedin a silica-treated dose and time-dependant manner, while HBE cells only showsilica-treated dose-dependant effect. In addition, we found a significantly decrease formiR-149in HBE cells after12hours treated with the150μg/ml of silica（P<0.000）,the expression of miR-149was almost totally inhibited by the silica exposure, whileno significance was found inA549cells after24hours treated with the50μg/ml ofsilica（P=0.234）. But a decreased trend can be found in it. At last, we verified that thedysregulated expression of miR-149with miR-149mimics and inhibitor transfectioninfluenced the IL-6expression in A549cells. High expression of miR-149inhibitedIL-6expression. Reverse results were found after miR-149was inhibited.Conclusion:（1） Inflammatory reaction was induced after silica exposure in mice;（2）Down regulation of miR-149and up regulation of IL-6may involved in theinflammation and fibrosis process of silica-induced pulmonary fibrosis;（3） miR-149can inhibit IL-6expression in A549cells.

          miR-149调控矽尘诱导肺纤维化的初步研究

缩略语5-7中文摘要7-11Abstract11-14第一部分：煤工尘肺特异的血清 miRNA 筛检15-35    前言15-17    调查内容与方法17-20    结果20-26    讨论26-30    参考文献30-35第二部分：miR-149 在 SiO_2粉尘所致肺纤维化中的功能学研究35-64    前言35-37    材料与方法37-50    结果50-58    讨论58-60    结论60-61    参考文献61-64综述64-79    参考文献72-79硕士期间发表论文79-97致谢97

  本文关键词：miR-149调控矽尘诱导肺纤维化的初步研究，由笔耕文化传播整理发布。