ciR-0000064/LRRC8D通路参与矽肺纤维化的研究

Occupational exposure to silica dusts occurs in many industries, giving rise to reduced lung function characterized by excessive fibroblast proliferation and collagen deposition and eventually leading to respiratory failure, which is a serious problem in developing and even developed countries. However, the exact etiology of silicosis is not well understood. Thus, further efforts are needed to find early molecular marker of diagnosis, new target to control the fibrosis induced by silica..Circular RNAs are ubiquitous in molecular biology. Recent studies have described the presence of circular RNA species from back-spliced exons in mammals (circular exonic RNAs, circRNAs) and have established that they are very abundant and are differentially expressed Circular RNAs may, for example, serve as transcription regulators or as sponges for small RNA regulators. .Our primary studies suggested that Leucine-rich Repeat-containing Protein 8d (LRRC8D) played a critical role in fibroblast proliferation and migration. However, the detailed cellular and molecular mechanisms underlying inflammation and the subsequent fibrosis in response to silica remain unknown. Circular RNA microarray assay suggested that ciR-0000064 level in mouse lung exposure to SiO2 was increased. Interestingly, ciR-0000064 is the regulator of miR-30, which have been show in our micro RNA microarray assay, in which we found that miR-30 can inhibit the expression of LRRC8D.. Therefore, we hypothesized that ciR-0000064 targeting regulation of LRRC8D via miR-30, the mechanism may involved in ceRNA. So ciR-0000064/ miR-30/LRRC8D pathway will be the potential target of curative treatment for silicosis involving in regulation of inflammation and fibrosis. The proposed studies 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, all of which are currently well established in our laboratory. This proposal is both novel and innovative in that the efficacy of regulation of ciR-0000064/ miR-30/LRRC8D can be of value to prevent or halt progression of silicosis in people. Our study will decipher the link between ciR-0000064/ miR-30/LRRC8D and inflammation with subsequent fibrosis, induced by silica providing a novel insight into the potential of ciR-0000064/ miR-30/LRRC8D in terms of opening up novel therapeutic avenues for silicosis.

长期吸入二氧化硅粉尘（SiO2）引起的肺组织纤维化的矽肺病影响患者的生活质量及预后。由于机制不明，临床面临早期缺少筛检方法，后期缺乏特效治疗的问题。环状RNA（circRNA，ciR）是新近确认的非编码RNA分子，可通过竞争性内源RNA(ceRNA)机制调控靶蛋白，具有结构稳定、丰度高和组织特异性表达等特征，可作为临床诊断标志物与疾病干预靶点的理想候选者。申请者前期ciRNA高通量筛选发现，ciR-0000064及其下游miR-30出现显著变化，其靶基因LRRC8D可能参与矽肺发生发展过程。本课题拟在前期工作基础上，以矽肺为研究对象，从ciR-0000064/ miR-30/LRRC8D可能参与SiO2诱导肺纤维化入手，通过构建矽肺动物和体外细胞模型，重点探讨及阐明ciR-0000064/ miR-30/LRRC8D通路对矽肺纤维化的作用及其机制，为矽肺早期诊断及治疗提供新思路和新靶点。

研究背景：长期吸入二氧化硅粉尘（SiO2）引起的肺组织纤维化的矽肺病影响患者的生活质量及预后。由于机制不明，临床面临早期缺少筛检方法，后期缺乏特效治疗的问题。环状RNA（circRNA，ciR）是新近确认的非编码RNA分子，可通过竞争性内源RNA(ceRNA)机制调控靶蛋白，具有结构稳定、丰度高和组织特异性表达等特征，可作为临床诊断标志物与疾病治疗靶点的理想候选者。本课题拟在前期工作基础上，重点探讨及阐明ciR-0000064/ miR-30/LRRC8D通路对矽肺纤维化的作用及其机制，为矽肺早期诊断及治疗提供新思路和新靶点。本项研究主要内容包括：1）临床水平观察ciR-0000064/miR-30/LRRC8D参与矽肺炎性反应和肺纤维化的研究；2）动物水平验证ciR-0000064/miR-30/LRRC8D参与矽肺炎性反应和肺纤维化的研究；3）细胞水平探究ciR-0000064/miR-30/LRRC8D参与矽肺炎性反应和肺纤维化的机制。重要结果及数据：1）临床标本上观察到circHDAC4（ciR-000064）在临床矽肺患者中出现显著变化，小鼠动物试验也验证了上述结果。2）LRRC8D在肺泡巨噬细胞中呈时间依赖性高表达；进一步研究发现上升的LRRC8D介导了SiO2引起的炎症促纤维化作用。3）为了进一步探讨LRRC8D参与矽肺进程的机制，结果发现circHDAC4通过吸附miR-30C，解除miR-30C对lrrc8d的抑制作用，进而导致LRRC8D表达升高；进一步功能学研究发现，circHDAC4对成纤维细胞的功能产生显著影响，进而明确ciR-0000064/miR-30/LRRC8D在矽肺炎症/纤维化进程中的作用。LRRC8D对下游细胞的影响可能通过自噬和泛素化机制发挥作用，因此对其LRRC8D下游重要功能分子HECTD1在ciR-0000064/miR-30/LRRC8D通路中可能参与的机制进行了进一步研究，结果发现，HECTD1通过而直接诱导成纤维细胞活化以及通过内皮间质转化参与矽肺纤维化进程。意义：本研究揭示了ciR-0000064/miR-30/LRRC8D通路在引起肺间质纤维化的发生和发展过程，这将为寻找新的矽肺治疗靶点提供新思路和理论依据。