LncRNA IRI-1调控Caspase-3在低温保护心肌缺血再灌注损伤中作用机制研究

Coronary heart disease is the main cardiovascular disease which increases the global burden of mortaily and morbidity. Reperfusion therapy, which promotes the rapid recovery of blood flow to the myocardial ischemic zone, may result in further complications such as irreversible tissue necrosis, which are collectively known as ischemic-reperfusion (I/R) injury. Reducing reperfusion injury has become one of the focuses of clinical research. Preliminary study showed: 1. Various of long non-coding RNAs (lncRNAs) are over expressed in mouse model with mild hypothermia (core body temperature 32°C) ischemia/reperfusion injury and lncRNA IRI-1 is the most obvious one. 2.The expression level of Caspase-3 is significantly reduced in cardiac myocytes in the hypothermia I/R mouse. 3. LncRNA IRI-1 locates in the nucleus of suckling mouse myocardial cells. Taken together, we suggest that abnormally up-regulated lncRNA IRI-1 inhibited the expression level of Caspase-3 and leading to the decreasing of apoptosis in cardiac myocytes in the hypothermia I/R mouse. This study will use the primary cultured suckling mouse myocardial cells and hypothermia I/R mouse model to study the mechanism of lncRNA IRI-1 regulating Caspase-3 by the technology of chromatin oligonucleotide precipitation (CHOP), RNA oligonucleotides chromosome conformation capture (R3C) and so on. We will explore the key factors of Caspase-3 down regulated by lncRNA IRI-1 and validate the function of the key factors, so as to provide theoretical basis for the cardioprotective effects of lncRNA IRI-1 against ischemic-reperfusion injury.

冠心病是主要的致死性心血管疾病，缺血再灌注损伤（IRI）是冠心病研究重点关注的问题。前期研究发现：（1）与常温相比，低温（32°C）缺血再灌注（I/R）模型鼠心肌中许多lncRNA表达上调，其中lncRNA IRI-1上调最明显（2）低温模型鼠心肌Caspase-3基因和蛋白表达明显降低（3）乳鼠心肌细胞中lncRNA IRI-1表达位于胞核，具备调控Caspase-3基因表达的空间条件。据此推测：低温下，异常升高的lncRNA IRI-1抑制Caspase-3表达，对抗I/R模型鼠心肌细胞凋亡。本研究将借助乳鼠心肌细胞和低温I/R小鼠模型，利用寡核苷酸沉淀（CHOP）、RNA染色体构象捕获（R3C）等技术，探究lncRNA IRI-1调控Caspase-3的关键中间因子及作用机制，并观察其对低温I/R模型鼠心肌的保护作用。该研究有望揭示低温下lncRNA IRI-1保护心肌的分子机制。

缺血再灌注损伤（IRI）是冠心病研究重点关注的问题。在心血管系统中，非编码RNA可调控细胞的分化、发育和凋亡等病理生理过程。本研究的主要研究内容：1）检测低温及缺氧后小鼠心肌细胞microRNA表达变化；2）分析LncRNA IRI-1相关microRNA在心梗中表达变化；3）验证心肌细胞凋亡相关的LncRNA IRI-1；4）敲除或过表达发现的关键microRNA，了解其在心肌细胞损伤中的作用。重要结果和科学意义：1）在缺氧和低温两种干预下收集心肌细胞RNA标本进行全基因组测序分析，结果发现LncRNA IRI-1相关miR-10b-5p、miR-19-1-5P、miR-483-3p等出现异常表达，进一步GO-pathway分析提示miR-10b-5p、miR-19-1-5P、miR-483-3p参与到炎症、缺氧信号通路、细胞代谢等病理生理过程中。2）于不同梗死时间后，收集相应梗死区域并检测其中LncRNA IRI-1相关micorRNA的表达情况，结果显示miR-10b-5p、miR-483-3p的表达水平于心梗后1和3天呈显著性下降，而在7天时则呈现反馈性升高，此外，体外培养小鼠原代心肌细胞在缺氧6小时后，miR-483-3p和miR-10b-5p表达呈显著性下降。由此表明microRNA-10b-5p、microRNA-483-3P可参与到心肌梗死病理生理过程中。3）miR-483-3p的模拟物（Mimic）和抑制物（Inhibitor），导入心肌细胞后并进行缺氧处理，发现导入miR-483-3p后，缺氧诱发的心肌凋亡可受到明显的抑制，而导入miR-483-3p抑制物则导致心肌细胞凋亡增多。此外，构建microRNA-483-3P慢病毒，构建小鼠心梗模型的同时心肌梗死周边组织注射miR-483-3P negative control以及overexpression慢病毒，一周后进行TTC染色发现miR-483-3p能够减少心肌梗死面积。4）原代心肌细胞转染miR-10b-5p negative control、mimic以及inhibitor 48h后，细胞缺氧3h，发现miR-10b-5p可抑制心肌细胞的凋亡。构建miR-10b-5p慢病毒亦发现类似现象。本课题的意义在于研究LncRNA IRI-1相关microRNA与心梗后心肌损伤之间的调控关系。