矽肺纤维化特异microRNAs和mRNAs标志物鉴定及其调控机制

The previous study of our research group maintained that microRNAs, after its transcription, regulate the gene expression of silicosis and play a critical role in silicosis fibrosis and that the research of its effect markers during-and-after the transcription in the initial diagnosis of silicosis remains to be conducted. This program, through occupation investigation combined with experimental methods, by adopting 1:3 nest design of case control and random contrast and applying cohort study, animal model and cell culture model, on the basis of 117 mRNAs and 19 microRNAs preliminarily sieved from the previous national foundation program, aims to investigate the regulation mechanism of mRNAs and microRNAs after their transcription by way of customized chips, quantitative screening, the verification of PicTar, TargetScan and qRT-PCR and the disturbance experiment of network regulation of excessive or insufficient gene expression of cell model once again. The program first compares the corresponding relationship between microRNAs in the sieved blood plasma and mRNAs and microRNAs in the cells of alveolar lavage fluid and peripheral blood, then determines the sensitivity and specificity of large sample by adopting dosage effect, and finally identifies the markers of mRNAs and microRNAs of silicosis. This program illustrates the regulation mechanism of silicosis in the perspective of after-transcription, which studies the transcriptive markers of the initial diagosis of silicosis in a groundbreaking way so as to have a bright outlook.

课题组前期研究认为转录后microRNAs调控矽肺表达基因并对纤维化起决定性作用，目前矽肺早期诊断的转录和转录后效应标志物研究尚属空白。本项目拟通过职业调查结合实验手段，采用1∶3巢式病例对照和随机对照设计，应用人群研究、动物模型和细胞培养模型，在上一国家基金初步筛选的117个mRNAs 和19个microRNAs的基础上，再次通过定制芯片定量筛选以及PicTar、TargetScan、qRT-PCR验证，结合细胞模型的基因敲低或过表达的网络调控扰动实验，探讨mRNAs和microRNAs转录后调控机制。通过比对筛选血浆microRNAs同肺泡灌洗液各细胞、外周血各细胞mRNAs 和microRNAs对应关系，应用剂量效应、大样本人群灵敏度特异度的判定，最终确定矽肺mRNAs 和microRNAs标志物。该项目从转录后水平阐明矽肺调控机制，创新性地研究矽肺早期诊断的转录标志物，前景广阔。

本项目应用人群研究、动物模型和细胞培养模型，通过比对筛选血浆microRNAs同外周血各细胞mRNAs 和microRNAs对应关系，应用剂量效应、大样本人群灵敏度特异度的判定，最终确定矽肺microRNAs标志物。通过芯片筛选以及qRT-PCR验证，结合细胞模型的基因敲低或过表达的网络调控扰动实验，探讨microRNAs转录后调控机制。.研究结果为：①血硅、尿硅对矽肺纤维化早期诊断有一定的指示意义，但血硅含量不能完全反映矽肺纤维化的程度。②矽肺早期诊断生物标志物血清miRNAs、外泌小体miRNAs、血清硅含量联合外泌小体microRNA均有较高的灵敏度和特异度，而血清硅含量联合外泌小体microRNA-16、let7d联合诊断灵敏度和特异度分别达97%和67%。③特异microRNAs联合矽尘调控肺泡上皮细胞间充质化机制不同。microRNA-200b均能促进正常和染尘肺泡上皮细胞间充质化；microRNA-483对正常和染尘肺泡上皮细胞均没有明确的间充质化作用；let-7d、miR-16、miR-150均抑制上皮细胞（正常和染尘）间质化的形成；microRNA-21、microRNA-29在无粉尘的条件下抑制上皮间充质化，在有粉尘刺激下促进肺泡上皮细胞间充质化；microRNA-448在无粉尘条件下对上皮细胞间充质化作用不明确，在粉尘条件下可促进肺泡上皮细胞间充质化。矽肺差异表达基因调控网络度大于5的关键节点为CCL7, GRN, IL-18, SFTPD, SPN, SPP1, TNF-α, TNFRSF。.该项目从转录后水平阐明矽肺调控机制，创新性地研究矽肺早期诊断的转录标志物，发表论文4篇，申请矽肺患者外周血外泌体早期诊断试剂盒发明专利一份，目前已在完善专利评审员的问题阶段，该发明专利进一步提高了矽肺患者早期诊断的灵敏度和特异度，应用前景广阔。