miR-221介导环形RNA_005647激活TGFβ1/TGFβR1-PAK1途径促进心肌纤维化

Cardiac fibrosis is an important character and cause of the diabetic cardiomyopathy, but the mechanism underlying the diabetic cardiac fibrosis has not been well elucidated. We observed that circular RNA circRNA_005647 was up-regulated in the myocardium of the diabetic mice. Over-expression of circRNA_005647 could significantly enhance the expressions of fibrosis-related genes in mouse myofibroblasts, and activated the TGFβ1/TGFβR1-PAK1 pathway.However, the molecular mechanism underlying the pro-fibrotic effect of circRNA_005647 is still unknown. The results of RNA pull-down and dual luciferase assay revealed the specific interaction between circRNA_005647 and miR-221-3p. We hypothesize that circRNA_005647 sponges miR-221-3p and derepresses the downstream target genes of miR-221-3p, including angiopoietin-like 2(ANGPTL2), TGFβ receptor type I(TGFβR1) and P21 Protein (Cdc42/Rac)-activated kinase 1(PAK1), contributing to the activation of TGFβ1/TGFβR1-PAK1 pathway and cardiac fibrosis. The transgenic circRNA_005647 mice and the wild type control mice, 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 cardiac fibrosis by circRNA_005647 at the whole organism,cellular and molecular level, respectively. The present study is expected to provide scientific evidence and material for developing new therapeutic approaches based on circular RNAs for treatment of diabetic cardiac fibrosis.

心肌纤维化是引发糖尿病性心肌病的最主要原因之一，但机制尚不明确。我们发现环形非编码RNA_005647(circRNA-5647)在糖尿病性小鼠心肌中表达增强，过表达circRNA-5647可促进小鼠心肌成纤维细胞中纤维化相关基因表达，并激活TGFβ1/TGFβR1-PAK1通路，但分子机制不明。RNA pull-down和双荧光素酶报告实验结果证实circRNA-5647与微小RNA miR-221-3p有特异结合作用。推测circRNA-5647通过解除miR-221-3p对靶基因ANGPTL2、TGFβR1和PAK1的抑制作用，激活TGFβ1/TGFβR1-PAK1通路，进而促发心肌纤维化。本项目拟建立circRNA-5647转基因小鼠，从整体-细胞-分子水平，阐明circRNA-5647在糖尿病性心肌纤维化中的作用机制，为进行基于环形非编码RNA的心肌纤维化治疗研究提供科学依据。

心肌纤维化是引发糖尿病性心肌病的最主要原因之一，但机制尚不明确。环形非编码RNA（circRNAs）是一类闭合环状的长链非编码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，不易受RNA外切酶影响，稳定性好。CircRNAs参与基因转录、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circRNA_005647及其宿主基因Myo9a在糖尿病db/db小鼠心肌和HG/Go诱导的小鼠CFs中，以及在Ang-II灌注诱导的小鼠心肌及Ang-II处理所诱导的小鼠心肌细胞中均表达增强。circRNA_005647主要存在于细胞质中，利用腺病毒介导过表达circRNA_005647可以特异抑制小鼠心肌成纤维细胞中纤维化相关基因(如Col1a1,Col3a1和Acta2)表达，抑制Ang-II诱导小鼠心肌细胞中心肌肥大相关基因(如Anp和Myh7)表达。机制研究表明：circRNA_005647可以分别特异结合小鼠心肌成纤维细胞和小鼠心肌细胞中的miR-27b-3p和miR-99b-5p；miR-27b-3p/Pparg轴介导circRNA_005647发挥抑制心肌纤维化的作用；miR-99b-5p通过Fgf21/Sod2轴介导circRNA_005647发挥抑制心肌细胞肥大作用。进一步建立心肌特异表达circRNA_005647的转基因小鼠（Tg-Myh6-circRNA_005647），检测结果证实，横主动脉缩窄诱导后Tg-Myh6-circRNA_005647小鼠的心肌纤维化和心脏功能受损程度减轻。本项目明确了circRNA_005647在发生纤维化的糖尿病小鼠心肌和Ang-II灌注诱导的小鼠心肌中表达升高，阐明了circRNA_005647抑制心肌肥厚和心肌纤维化的分子机制，为开展基于circRNA_005647为干预靶点的心肌重构的治疗研究提供了科学依据和资料。