环形非编码RNA circRNA_000203促进衰老性心肌重构的机制研究

Cardiac remodeling is an important character of the aged heart, but the molecular mechanism of the aging-associated cardiac remodeling has not been well elucidated.The increasing evidence shows that non-coding RNAs participate in the pathology of the aging-associated cardiac remodeling. We observed that circular RNA circRNA_000203 was up-regulated in the myocardium of D-galactose–induced aged mouse. Over-expression of circRNA_000203 could significantly enhance the hypertrophic phenotype in mouse cardiomyocytes and enhance the fibrotic phenotype in mouse myofibroblasts, respectively. The results of dual luciferase assay and RNA pull-down assay revealed the specific interaction between circRNA_000203 and miR-26b. We hypothesize that circRNA_000203 sponges miR-26b and derepresses the downstream target genes of miR-26b, including Col1a2、CTGF、MEF2C、CCND1，-2，CCNE1 and CDK6, contributing to cardiac fibrosis and cardiac hypertrophy. The transgenic circRNA_000203 mice and the wild type control mice, and neonatal mouse cardiomyocytes and cardiac fibroblasts derived from the above two kinds of mice will be utilized in the present project. We will demonstrate the mechanism underlying the enhancement of the aging-associated cardiac remodeling by circRNA_000203 at the whole organism,cultural vessel,cellular and molecular level, respectively. The present study is expected to provide scientific evidence and material for developing new therapeutic approaches based on circRNAs for the aging-associated cardiac remodeling.

心肌重构是衰老心脏的重要特征，但衰老性心肌重构的机制不明。越来越多的证据显示非编码RNA(ncRNA)参与衰老性心肌重构的病理过程。我们证实环形RNA circRNA_000203（circRNA-203）在D-半乳糖诱导衰老小鼠心肌中表达上调，过表达circRNA-203能显著增强小鼠心肌细胞和心肌成纤维细胞的肥大表型和纤维化表型。双荧光素酶报告实验和RNA pull-down结果证实circRNA-203与微小RNA miR-26b有特异结合作用。推测circRNA-203通过特异结合miR-26b来解除其对靶基因Col1a2、CTGF、MEF2C、CCND1，-2，CCNE1和CDK6的抑制作用，进而促发心肌重构。拟建立circRNA-203转基因小鼠，从整体-细胞-分子水平，阐明circRNA-203作用的分子机制，为开辟以环形RNA为靶点的衰老性心肌重构治疗研究提供科学依据。

心肌重构是衰老心脏的重要特征，但衰老性心肌重构的机制不明。环形非编码RNA（circRNAs）是一类内源性非编码RNA，主要来源于编码基因外显子或内含子区，circRNAs不含3′或 5′末端，呈闭合环状结构，不受RNA外切酶影响，稳定性好、不易降解。CircRNAs参与了转录、mRNA剪接、RNA降解和翻译等生物学过程，且与心血管病等多种人类疾病的发生发展密切相关。通过该项目研究，明确了来自于宿主基因Myo9a的circRNA_000203在糖尿病小鼠心肌、增龄衰老性小鼠心肌和Ang-II灌注小鼠心肌中表达增强，在Ang-II诱导的小鼠心肌细胞和心肌成纤维细胞中表达也明显升高。circRNA_000203主要存在于细胞质中，NF-kB信号介导了Ang-II诱导的小鼠心肌细胞中circRNA_000203表达升高。利用腺病毒介导过表达circRNA_000203可以特异增强小鼠心肌细胞中肥厚相关基因Anp和Myh7表达，增加心肌细胞面积；过表达circRNA_000203能增强小鼠心肌成纤维细胞中纤维化相关基因Col1a1, Col3a1和Acta2表达，表明circRNA_000203具有促进心肌肥厚和心肌纤维化的作用。机制研究表明：circRNA_000203通过特异结合miR-26b-5p靶向增加Ctgf和Col1a2表达发挥促心肌纤维化作用，circRNA_000203通过特异结合miR-26b-5p、miR-140-3p靶向增强GATA4表达发挥促心肌肥厚作用。进一步建立心肌特异表达circRNA_000203的转基因小鼠（Tg-Myh6-circRNA_000203），检测结果证实，Tg-Myh6-circRNA_000203小鼠心肌中circRNA_000203升高20多倍，miR-26b-5p和miR-140-3p表达降低，GATA4表达升高；Ang-II灌注诱导后Tg-Myh6-circRNA_000203小鼠的心脏功能受损加重，心肌肥厚和纤维化程度增加。本项目明确了circRNA_000203在衰老性小鼠心肌中表达升高的调控机制，阐明了circRNA_000203促进心肌肥厚和心肌纤维化的分子机制，为开展基于circRNA_000203为干预靶点的衰老性心肌重构的治疗研究提供了科学依据和资料。