血清microRNA作为煤工尘肺生物标志物及其机制的研究

Coal workers' pneumoconiosis (CWP) is one of the most severe occupational diseases in China, which encloses coal worker's lung, silicosis and anthraco-silicosis without effective treatments. The anthraco-silicosis is the most common in CWP, in which free silica is the main cause for lung fibrosis. CWP diagnosis mainly depends on X-ray chest film and the history of occupational dust exposure, still lacking the sensitive and specific serological biomarker to make early diagnosis. MicroRNAs (miRNAs)，as the post-transcriptional gene expression regulators, are very stable in the blood. Serum miRNAs are from blood cells in normal. However, they can be produced by the pathologic tissues and released into the blood in abnormality. Recently, some circulating miRNAs in the blood have been successfully revealed as the biomarkers for several diseases. Multiple cytokine networks and related genes which are partly regulated by miRNAs involved in the development of silicosis.The main purpose of the project aims to find the specific miRNAs as the biomarkers of CWP or silicosis. The non-coding RNAs are detected by high-flux sequencing, and bioinformatics are taken to identify the different miRNAs expressions in serum between normal and CWP patients, their possible target genes and the regulating signal pathways. The possible related miRNAs are selected and will be preliminarily validated by the case-control study. The high-flux array and bioinformatics are used to screen different expression of miRNA and cytokines in lung tissues during the development of mouse silicosis. The possible silicosis related miRNA, their target genes and cytokines will be selected and validated by animal models. Combined preliminary results from population and animal study and carry out the secondary validation of serum miRNAs which provide basis for further studies on the molecular mechanisms by cell models. Finally silicosis related miRNA and their target genes will be identified by aggregate analysis of the whole data. These may provide the reliable theory basis for early diagnose and new target treatment for silicosis or CWP.

煤工尘肺是我国最严重的职业病之一，诊断主要依赖高仟伏胸片及粉尘接触史，缺少辅助早期诊断的特异性血清标志物。该病的发生发展涉及大量细胞因子及其相关基因的复杂网络调控，miRNA在其中起着重要的作用。血清miRNA正常状态下主要来自于血细胞，在疾病状态下部分来自于病变组织。本项目采用高通量测序及芯片技术检测血清ncRNA和小鼠肺组织miRNA及细胞因子表达，用生物信息学技术筛检接尘正常人与煤工尘肺病人、正常和矽肺小鼠肺组织表达差异的miRNA、可能作用的靶标及调控的信号通路，结合细胞因子筛检结果，选择有意义miRNA及其靶标和细胞因子分别进行人群和动物组织验证；根据人群和动物实验结果进行再次人群验证；细胞模型探讨miRNA的作用机制。综合人群、动物和细胞实验结果确定煤工尘肺相关的miRNA及其靶基因，探讨其在尘肺发生发展中可能的分子机制，为尘肺病早期诊断及寻找新的治疗靶点提供可靠的理论。

煤工尘肺包括矽肺、煤矽肺和煤肺三种，以煤矽肺最为常见。是我国最多见、最严重的职业病之一，尘肺病的发病机制尚未完全阐明，目前无特效治疗方法，因此探明矽尘诱导肺纤维化的分子机制，对于矽肺的预防、诊断和治疗方法研究均十分重要。大量研究表明尘肺病的重要病理特征为成纤维细胞过度增殖，成纤维细胞向肌成纤维细胞分化以及细胞外基质的过度沉积，其发生发展过程中涉及大量细胞因子及其相关基因的复杂网络调控，miRNA在其中起着重要的作用。血清miRNA正常状态下主要来自于血细胞，在疾病状态下部分来自于病变组织。本项目采用高通量测序及芯片技术检测血清ncRNA和小鼠肺组织miRNA及细胞因子表达，用生物信息学技术筛检接尘正常人与煤工尘肺病人、正常和矽肺小鼠肺组织表达差异的miRNA、可能作用的靶标及调控的信号通路，选择有意义miRNA进行动物和细胞实验验证，运用细胞模型进行发病机制的研究，确定尘肺病人特异的多条miRNAs、作用的靶基因及参与主要调控的信号通路：（1）miR-486-5p通过靶向调控TGF-β信号通路中关键的信号分子SMAD2和Col VI影响矽尘诱导的肺纤维化发生与发展；（2）miR-449a负调控Bcl2的表达，影响了肺成纤维细胞的自噬水平，从而影响了肺纤维化的发生发展；（3）miR-449a负调控NOTCH1的表达，影响下游分子Snail的表达，从而抑制了上皮细胞间质转化；（4）miR-503-5p可以通过抑制PI3K p85以及其下游的p-AKT/Snail的表达来抑制上皮细胞间质转化；（5）miR-149可负调控细胞IL-6的表达水平影响矽肺的发生及发展。同时探讨这些miRNA作为干预剂在矽肺发生发展中的作用，也初步探讨部分miRNAs在尘肺病患者血清中的表达水平。上述结果为揭示矽肺发病的分子机制提供了新的线索，也为矽肺病新的治疗靶点提供了可靠的理论依据。