circRNA-mus 调控miRNA-30c/CTGF对失神经支配喉肌萎缩纤维化的作用

Progressive atrophy and fibrosis induced by denervation severely impairs the structural and functional integrity of intrinsic laryngeal muscles, resulting in the deterioration of the regenerative environment. Articles and our previous investigation have demonstrated that TGFβ1/Smads signaling plays a pivotal role in the progress of atrophy and fibrosis in denervated muscles, in which CTGF is a key pro-fibrotic effector in downstream cascades.However, blocking TGFβ1 as a target will be show a significant side effects.Further study revealed that miRNA-30c can specifically target CTGF at the transcriptional level in skeletal muscles. Recent study reported that a new kind of non-coding circle RNA (circRNA) modulates gene expression via combining with specific miRNAs.These circRNAs exhibit important effects in the physiopathological process, including myocardial fibrosis.But the underlying mechanism remains unclear. Our preliminary experiment found that a new circRNA was specifically up-regulated in the denervated laryngeal muscles in mice. The detailed gene sequence of this circRNA was detected by high-throughout RNA sequencing. This new circRNA doesn’t match the sequence of any reported circRNAs in the established circ-base bank, thus we temporarily named it as circRNA-mus. Bioinformatics analysis predicted circRNA-mus may directly target miRNA-30c because it abundantly contains the combining sites of miRNA-30c.As the literature shows that circRNA can bind to miRNA to regulate CTGF in myocardial fibrosis. And our further experiments confirmed miRNA-30c could be inhibitted by circRNA-mus.We hypothesized that circRNA-mus may directly combine with miRNA-30c to regulate the expression of CTGF, by which circRNA-mus modulates the atrophic and fibrotic process in denervated laryngeal muscles. For this purpose, we will establish a mice model of recurrent laryngeal nerve paralysis and TGFβ1-induced myofibroblast transition of laryngeal myofibers. In vivo and in vitro experiments are designed to demonstrate the spatio-temporal expression alterations of circRNA-mus, miRNA-30c and CTGF, and elucidate the mechanisms of circRNA-mus regulating CTGF via targeting miRNA-30c. This study is expected to explore a new therapeutic target that potentially prevents or alleviates the atrophic and fibrotic process, and maintains the regenerative capacity of the denervated laryngeal muscles. The better laryngeal function may be regained after laryngeal reinnervation through a novel therapeutic strategy targeting circRNA-mus.

失神经喉肌萎缩纤维化是导致喉神经修复效果欠佳的重要原因。文献及申请人证实TGFβ1/Smads在转录水平调控CTGF在失神经肌肉萎缩纤维化中的作用，但以TGFβ1为靶点加以干预机体副反应大。已证实miRNA30c可在转录后水平调控CTGF。我们发现circRNAmus在失神经喉肌中高表达，生物信息学分析其序列内富有miRNA30c的结合位点；文献显示circRNA可结合miRNA调控CTGF发挥心肌纤维化作用，结合我们的前期实验，推测circRNAmus可结合miRNA30c调控CTGF，参与失神经喉肌萎缩纤维化进程。为此本项目采用基因干预、原位杂交、PCR、蛋白印迹、AGO免疫共沉淀、荧光素酶报告基因等技术，从基因、分子、细胞和组织层面，阐明circRNAmus调控miRNA30c/CTGF对失神经喉肌萎缩纤维化的作用及机制。旨在为寻找延缓失神经支配喉肌萎缩纤维化新靶点提供新理论新思路。

失神经喉肌萎缩纤维化是导致喉神经修复效果欠佳的重要原因，具体机制仍不明确。本课题组发现在失神经骨骼肌萎缩纤维化过程中，circRNA mus的表达显著升高，而miRNA-30c的表达明显下调。为明确导致miRNA-30c低表达的因素，通过全基因测序以及生物信息学分析，我们筛选出circRNA mus富含miRNA-30c的结合位点，通过干预调节circRNA mus在细胞中的基础表达，发现过表达circRNA mus可显著抑制miRNA-30c，而抑制circRNA mus则呈相反的趋势。提示在失神经骨骼肌萎缩纤维化过程中circRNA mus的表达丰度升高可能是导致miRNA-30c下调的主要原因之一。我们采用基因干预、PCR、蛋白印迹、荧光素酶报告基因等技术在体和离体实验中进一步研究了miRNA-30c及其调控纤维化的重要分子靶点CTCF。证实了miRNA-30c的干预可显著削弱circRNA mus对纤维化的调控作用。circRNA mus显著升高的情况下，通过海绵样吸附作用抑制了miRNA-30c的活性，导致其靶点CTCF的转录激活，从而促使纤维化相关的分子如a-SMA、羟脯氨酸的表达以及含量的增加，最终诱导纤维化的形成。这为寻找延缓失神经支配喉肌萎缩纤维化新靶点提供了新理论新思路。