MiR-181c对心脏室间隔缺损发生中心肌细胞功能紊乱的作用机制研究

The pathogenesis of ventricular septal defect (VSD) is still unclear. Our early study found that the expression of miR -181c in myocardial tissues of VSD patients rose abnormally and significantly inhibit the the expression of its target gene, BMPR2. BMPR2 is an important receptor of BMP signaling pathways, mainly regulating the downstream Smad signaling pathways and participating in the arrangement of actin.Through overexpressing miR-181c, we observed obviously disordered arrangement of myocardial cytoskeleton. So, we speculate that the regulation of miR-181c to BMPR2 could be the important molecular mechanism of the VSD occurrence and development. Then we introduced BMPR2 to the myocardial cells and observed that it could save the myocardial cytoskeleton's abnormal arrangement. However, how it acts on the cytoskeleton and its regulation mechanism to the downstream signal is unclear yet. Therefore, we proposed to carry out the following research:1) Explore the influence of miR - 181 c on BMPR2 LIMK - Cofilin signaling pathways and BMPR2 - Smad1/5/8 - Erk1/2 signaling pathways; 2) Using human IPS technology to study whether miR-181c is the leading cause of VSD; 3) Manipulate the expression of miR - 18 c to observe whether it could improve patients' myocardial cell dysplasia.

本课题前期研究发现，VSD患者心肌组织中miR-181c表达异常升高，并显著抑制其靶基因BMPR2的表达。BMPR2是重要的BMP信号通路受体，主要调控下游Smad信号通路及参与肌动蛋白的排列。我们将miR-181c导入心肌细胞，发现其过表达可明显造成心肌细胞骨架排列紊乱;若同时将BMPR2导入心肌细胞发现可补救前者引起的心肌骨架异常。我们推测，心肌细胞中miR-181c对BMPR2的调控可能是致VSD发生的重要机制。然而其如何作用于细胞骨架以及其对下游细胞信号的调控机制仍未明确。因此，我们拟开展以下研究：1）探讨miR-181c对BMPR2-LIMK-Cofilin及BMPR2-Smad1/5/8-Erk1/2信号通路的影响；2）利用VSD患者IPS细胞模型研究miR-181c是否是导致病人心肌细胞功能紊乱的因素；3）操控miR-18c的表达观察能否改善患者心肌细胞发育异常。

本课题旨在探讨miR-181c对BMPR2及其下游信号通路的调控作用，阐释其如何作用于心肌细胞骨架及对室间隔缺损中的调控机制。结果显示：1）原代大鼠心肌细胞中导入miR-181c mimic后，心肌细胞凋亡增加，增殖减少；显微镜下观察到心肌细胞骨架排列明显紊乱。2）原代大鼠心肌细胞中操纵miR-181c的表达水平，可显著影响LIMK/Cofilin蛋白的磷酸化水平及Smad1/5/8复合体的形成和Erk1/2的磷酸化水平。同时荧光显微镜下观察到心肌细胞骨架结构和分布也发生改变。这一变化可被BMPR2部分挽救。3）与对照组相比，室间隔缺损患者IPS源性心肌细胞中miR-181c表达水平升高，且存在心肌细胞骨架结构排列紊乱现象；当导入 BMPR2-LIMK-Cofilin 信号通路或 BMPR2-Smad1/5/8-Erk1/2 信号通路抑制剂时，可部分纠正心肌细胞表型异常。研究结果表明miR-181c 可能通过调控BMPR2-LIMK-Cofilin及BMPR2-Smad1/5/8-Erk1/2信号通路磷酸化水平，影响心肌细胞骨架排列，进而在心脏形成早期引发心脏结构缺陷，最终导致室间隔缺损的发生。本研究利用原代大鼠心肌细胞及室间隔缺损患者 IPS 干细胞作为细胞模型，验证 miR-181c 对室间隔缺损患者心肌细胞功能紊乱的影响，可揭示miR-181c 对室间隔缺损的致病机制，同时也为将来在体使用 miRNA 干预提供重要的理论基础和实践数据。