骨髓增殖性肿瘤JAK2V617F转基因小鼠骨髓细胞外泌体转运miR-143在心脏肥大中的作用及机制

Myeloproliferative neoplasms (MPNs) represent a range of incurable haematological malignancies, but the mortality of MPNs can be reduced by retarding and decreasing the patients’ cardiovascular complications. The mutation of JAK2V617F is an important molecular mechanism in the pathogenesis of MPNs. Our early research showed the cardiac hypertrophy in JAK2V617F mice were caused by abnormal matrix protein synthesis and degradation in fibroblasts, and that belonged to the remote control. Recently, some research found that MiR-143 expression was positively related to JAK2V617F and MPNs clinical phenotype, and miR-143 could regulate the expression and activation of matrix metalloproteinase (MMPs). The exosome is an important medium to transfer microRNA signal molecules in regulating function of cardiac fibroblast. Therefore, we put forward the hypothesis that in JAK2V617F transgenic mice，tumor cells in bone marrow secret and transship miR-143 by exosomes to cardiac fibroblast to regulate the activity of MMPs, promote fibroblasts proliferation and collagen secretion, and then mediated cardiac hypertrophy. In order to prove the hypothesis, firstly, the expressions of miR-143 in exosomes will be detected by in situ hybridization and Real-time PCR. Secondly, we will test the effect of miR-143-inhibitor on cardiac hypertrophy mediated by lentivirus to demonstrate the role of miR-143 on cardiac hypertrophy in JAK2V617F transgenic mice. Lastly, we will confirm the role of exosomes transshipping miR-143 to cardiac fibroblast on inhibition of MMPs activity, promoting fibroblasts proliferation, collagen secretion and cardiac hypertrophy in vivo and in vitro. The successful implementation of the project will provide new ideas and new targets for prevention and treatment of MPNs and relative complications.

骨髓增殖性肿瘤(MPNs)是一类恶性血液病，但心血管事件是主要死因。JAK2V617F突变是MPNs发生的重要机制。我们的前期研究显示JAK2V617F转基因鼠心脏肥大的机制是成纤维细胞功能异常，属于远距离调控。有研究发现miR-143表达与JAK2V617F突变率及临床表型正相关，且miR-143能够调控基质金属蛋白酶(MMPs)的功能。外泌体是传递microRNA信号分子的介质。因此JAK2V617F转基因小鼠骨髓细胞可能通过外泌体转运miR-143至心脏，抑制MMPs功能，引起成纤维细胞功能异常导致心脏肥大。本项目通过原位杂交和PCR检测外泌体中miR-143表达；慢病毒介导miR-143-inhibitor研究miR-143在转基因小鼠心脏肥大中的作用；体内外实验论证外泌体转运miR-143对MMPs活性及心肌的影响。本项目成功实施将为MPNs及其并发症的防治提供新靶点。

心肌肥大是对过度血液动力学负担或病理状态的一种补偿生理反应，目前尚没有特定的药物可用于治疗心脏肥大。本项目完成了在心肌肥大过程中miR-143-3p/ERK5表达水平对心肌肥大及炎症相关通路影响的检测；同时，完成了阿托伐他汀治疗心肌肥大过程中AMPK变化以及对下游miR-143-3p影响的检测及Foxo1/miR-143-3p与下游Smad2相关纤维化过程变化的检测。本研究首先在体内通过主动脉弓缩窄法（TAC）建立心肌肥大大鼠模型，同时在体外利用H2O2干预H9C2细胞诱导心肌肥大氧化应激模型，检测心脏组织和H9C2细胞中miR-143-3p及部分通路中关键分子的表达；此外，通过研究miR-143-3p与炎症及氧化应激的相关性阐明miR-143-3p在心肌肥大中的作用。其次，通过体内和体外实验分别建立心肌肥大大鼠模型和Ang II诱导的H9C2细胞肥大特征模型，然后应用阿托伐他汀治疗动物和细胞，评估对心脏重量和结构以及细胞生存力、表面积和凋亡的影响；进一步探讨阿托伐他汀通过影响AMPK/Foxo1/miR-143-3p轴发挥抗心肌肥大作用。再次，通过体内建立心肌肥大大鼠模型和体外建立TGF-β1诱导的CFs细胞纤维化特征模型，检测阿托伐他汀对心脏纤维化发展的影响；在此基础上分离心脏成纤维细胞，同时对TGF-β1、Smad2、p-Smad2、Smad3、p-Smad3进行检测，评估miR-143-3p过表达对阿托伐他汀抗纤维化功能的影响，从而阐明阿托伐他汀通过抗纤维化治疗心肌肥大的分子制。最后，综合以上内容揭示阿托伐他汀治疗心肌肥大的内在机制，对开发心肌肥大治疗新靶点，有着积极的指导作用。项目支持下已发表SCI文章4篇。该项目的成果后续研究获得吉林省科技厅自然科学基金资助。该项目协助培养博士研究生1名，硕士研究生1名。