microRNA-103-3p心肌靶向表达通过下调TLR4改善心梗后慢性心力衰竭的研究

Chronic heart failure is characterized by cardiac inflammation, which is usually a manifestation of innate immune response or occasionally a combination of innate and adaptive immunity. Toll-like receptors (TLRs) are key molecules involved in innate immunity. At least 11 members of the TLR family have been identified in mammals. We previously reported that TLR4 mediates cardiac inflammation induced by myocardial infarction, and others reported that TLR4 is involved in the pathogenesis of CHF. The upstream mechanisms that control TLR4 expression and/or activity remain unknown. We addressed this issue by studying microRNAs, a class of short, single-stranded RNAs that suppress protein expression through annealing with complementary sequences in mRNAs. In the pilot experiments, we observed that miR-103-3p transfection suppressed the expression of the luciferase reporter containing the 3'-untranslated region (3'-UTR) of TLR4 mRNA. Furthermore, we observed decreased expression of miR-103-3p and increased expression of TLR4 in both infarct and noninfarct areas following coronary occlusion in mice. We hereby propose that miR-103-3p overexpression can downregulate TLR4 and improve CHF after myocardial infarction. This project will innovatively apply the technique of ultrasound-targeted microbubble destruction (UTMD) to obtain a high-specific, high-efficient and long-lasting overexpression of miR-103-3p in the heart of intact mice. Using this miR-103-3p overexpression model, we will observe whether TLR4 expression and its signaling activity are downregulated, and whether cardiac inflammation and CHF are improved after myocardial infarction. Additionally, TLR4-overexpressing and TLR4 knockout mice will be used to verify whether TLR4 mediates CHF and whether miR-103-3p overexpression improves CHF through suppressing TLR4. This project will provide novel insights into the miRNA molecules that might be of therapeutic benefit for CHF, and develop a novel application of UTMD to transfect miRNA as a target therapy for heart diseases.

慢性心力衰竭（CHF）的一个重要病理特征是心肌炎症反应。我们报道toll样受体-4（TLR4）介导缺血心肌炎症反应，文献报道TLR4参与心梗后CHF，但TLR4的上游调控机制不明。microRNA是能够从转录后水平抑制基因表达的一类小分子。我们预实验发现miR-103-3p在体外可抑制TLR4表达，心梗后CHF心肌TLR4表达高而miR-103-3p表达低。基于上述研究，本课题提出miR-103-3p高表达能够下调TLR4通路而改善心梗后CHF这一新设想。拟创新性应用超声靶向微泡破碎技术（原理是声穿孔效应）使小鼠心肌特异性、稳定、持续高表达miR-103-3p，在此模型研究心梗后TLR4表达及通路活性是否下调、心肌炎症反应和CHF是否减轻。并且，利用TLR4过表达和基因敲除小鼠，证明miR-103-3p改善CHF机制在于下调TLR4。本课题将为研究治疗CHF的分子靶点和靶向策略提供新资料。

慢性心力衰竭（CHF）的一个重要病理特征是心肌炎症反应。我们前期报道toll样受体-4（TLR4）介导缺血心肌炎症反应，文献报道TLR4参与心梗后CHF，但TLR4的上游调控机制不明。microRNA是一类从转录后水平抑制基因表达的小分子。我们预实验发现miR-103-3p在体外能够抑制TLR4表达，心梗后CHF心肌TLR4表达高而miR-103-3p表达低。基于上述研究，我们提出了miR-103-3p高表达能够下调TLR4通路而改善心梗后CHF这一新设想。本项目通过永久性结扎小鼠冠状动脉左前降支制备心梗模型，监测了心梗后CHF进程中miR-103-3p和TLR4表达变化，观察到miR-103-3p表达减少，而TLR4表达增加，且TLR4增加的细胞来源主要为心肌细胞；我们应用了超声靶向微泡破碎（UTMD）这一新型基因转染技术，利用声穿孔效应导入miR-103-3p表达载体，使活体小鼠心肌特异性、稳定、持续高表达miR-103-3p；在此模型以及培养的心肌细胞模型，研究了miR-103-3p高表达对TLR4表达及其通路活性的抑制作用，以及对心肌炎症反应、心肌重塑和心功能的改善作用；并且，通过shRNA慢病毒载体特异性干预TLR4，以及应用TLR4基因敲除小鼠，验证了miR-103-3p改善心梗后CHF的机制在于下调TLR4这一设想。本项目首次应用UTMD技术给小鼠心肌组织转染miR-103-3p慢病毒表达载体，具有靶向性强、表达效率高、无创、能够反复实施等优点，对于开发UTMD新的应用在技术方面具有开拓性。本项目研究已在SCI期刊发表论著2篇、综述1篇，还有部分研究结果将在后续发表，为认识CHF的发生机制以及寻找潜在的治疗靶点和靶向策略提供了新资料。