lncRNA-H19/miR-29b-3p/BBC3通路调控矽肺纤维化的机制研究

The inhalation of silicon dioxide particles leads to silicosis, an untreatable respiratory disease characterized by progressive pulmonary fibrosis. Because of the highest morbidity, disability and mortality rate, the harmfulness of silicosis occupied the first place among pneumoconiosis in China. Although substantial and increasing studies were undertaken to dissect the pathological mechanisms underlying the process of pulmonary fibrosis induced by silica, the primary cause of this devastating pathophysiological process remains elusive. On the other hand, although comprehensive management strategies help to improve quality of life and slow deterioration, there is no targeted curative treatment exists. Thus, further efforts are needed to find new target to control the fibrosis induced by silica. Recent literature have described the presence of long noncoding RNA (lncRNA) in different physiological and pathological process. Because of the specific space structure, lncRNA could modulat the expression of numerous downstream protein via different mechanisms, such as competing endogenous RNA (ceRNA), in which, lncRNA act as sponges for small RNA. Our previous studies suggested that BBC3/PUMA, an important member of apoptosis protein family, play a key role in fibroblast proliferation and migration induced by silica, following acceleration of pulmonary fibrosis. However, the detailed cellular and molecular mechanisms of BBC3/PUMA underlying the pulmonary fibrosis triggered by silica remain unknown. LncRNA microarray assay suggested that lncRNA-H19 expression in peripheral plasma from patients of silicosis was increased compared that of healthy donor. Bioinformatics analysis indicate lncRNA-H19 is the regulator of BBC3/PUMA. Furthermore, microRNA microarray assay found that miR-29, which can be regulated by LncRNA-H19, was also decreased. Interestingly, miR-29 can also inhibit the expression of BBC3/PUMA. Based upon our previous study and primary data from our lab, we hypothesized that lncRNA-H19/miR-29/BBC3 pathway will be the potential target of curative treatment for pulmonary fibrosis induced by silica. The proposed study will be initiated in human sample followed by study in intact animals as well as primary cell cultures. Studies using classic pharmacological methods will be combined with molecular biological techniques as well as immunological methods. Our study will decipher the link between lncRNA-H19/miR-29/BBC3 pathway and pulmonary fibrosis induced by silica, providing a novel potential target from lncRNA to open up novel therapeutic avenues for silicosis.

矽肺是吸入含SiO2粉尘引起以肺纤维化为特征的的严重呼吸病，尚无特效治疗措施，其主因在于矽肺纤维化的发病机制还未明确，缺乏针对性干预治疗的靶点。长链非编码RNA(LncRNA)可通过ceRNA等多种机制调控下游蛋白表达。申请者前期研究发现，凋亡蛋白BBC3(PUMA)参与了矽肺纤维化的发生发展；miRNA高通量筛选发现miRNA-29b-3p参与下调BBC3；LncRNA高通量筛选提示lncRNA-H19具有与miRNA-29b-3p的潜在结合位点；生物信息学分析提示BBC3是受lncRNA-H19调控的宿主基因。据此，申请者提出“lncRNA-H19/miRNA-29b-3p/BBC3通路是矽肺纤维化诊治的有效和关键靶点”假说，拟应用分子生物学和经典药理学方法，从整体、细胞和分子水平系统阐明此通路在矽肺纤维化中的功能和作用机制，为寻找临床干预治疗矽肺纤维化的靶点提供新思路和实验依据。

矽肺是一种因长期吸入二氧化硅粉尘导致的肺纤维化疾病。作为一种危害严重的职业性肺病，矽肺因其高发病率、致残率和致死率一直被公共卫生研究领域所关注，且迄今缺乏特效治疗手段。矽肺无有效疗法主要原因是缺乏疾病早期诊断的生物标志物和疾病进展后有效的抗肺纤维化治疗方法，因此，探索其发生发展过程中的效应细胞和分子的功能变化及其调控机制，具有重要意义。环状RNA（circRNA）是一类新的非编码RNA，作为一种转录调节因子或小RNA分子海绵的独特调控作用，已被证明在矽肺病中具有重要作用。在本研究中，我们探讨了经SiO2处理后的肺成纤维细胞中，circHECTD1/HECTD1（含E3泛素蛋白连接酶1的结构域）的表达，及其对细胞活化导致的肺纤维化病变的调控作用及其机制。试验中，我们主要采用原代人肺成纤维细胞（HPF-a）为研究对象，应用实时定量PCR（qRT-PCR），WB（western blot），荧光原位杂交（FISH）检测mRNA，HECTD1蛋白质，和circRNA等的表达水平；应用LC3B-LV-RFP慢病毒转染检测评估细胞自噬在此过程中的作用；应用CRISPR/Cas9系统特异性敲除HECTD1，结合MTT、BrdU和迁移实验，探讨SiO2诱导后的细胞功能变化。试验结果发现：经SiO2处理后，HPF-a细胞中的circHECTD1表达降低与同时期HECTD1的表达升高具有关联性。SiO2诱导产生的细胞自噬可被HPF-a细胞的circHECTD1高表达或HECTD1的敲降所逆转。在HPF-a细胞中过表达或敲降HECTD1，可通过下游自噬作用，参与调控SiO2诱导的成纤维细胞的活化、增殖和迁移。暴露于SiO2的矽肺小鼠动物模型试验，证实了HECTD1存在于肺成纤维细胞。本课题试验预期目的达到，发现了circHECTD1/HECTD1轴与经SiO2诱导的成纤维细胞的活化及其后继的肺纤维化之间存在联系，circHECTD1/HECTD1有望作为潜在靶标，为未来矽肺早期诊断和抗纤维化药物研发提供了新思路和试验基础。围绕本课题，目前已发表SCI论文1篇和国内核心期刊论文2篇，培养硕士研究生1名,于2019年顺利毕业。