LncRNA MIAT调控Cryab参与深低温低流量后脑缺血再灌注损伤

Cerebral ischemia-reperfusion injury after deep hypothermic low flow(DHLF) is an urgent issue for complex congenital heart disease surgery. Recent studies have shown that LncRNA plays an important role in neurological damage. In this study, LncRNA sequencing showed that LncRNA MIAT(MIAT) was highly expressed in the cerebral cortex after DHLF. MIAT was widely involved in the regulation of apoptosis, and neuronal apoptosis was the main manifestation of brain injury after DHLF, suggesting that MIAT may be involved in cerebral ischemia-reperfusion injury after DHLF. Bioinformatics analysis and pre-experimentation showed Cryab was the key target of MIAT. Meanwhile, Cryab plays an important role in anti-apoptosis. Accordingly, we hypothesized that LncRNA MIAT regulates Cryab gene to involve in cerebral ischemia-reperfusion injury after DHLF. In this study, we planned to build the DHLF cell and animal models. The overexpression and silencing strategies were used. The relationship between MIAT and Cryab was verified by RIP and RNA pull-down assay, which can explain the mechanism of cerebral ischemia-reperfusion injury after DHLF. The solution of this scientific problem will provide a new theoretical basis for brain protection strategy after DHLF.

深低温低流量(DHLF)后脑缺血再灌注损伤是目前复杂先心病体外循环术后亟待解决的问题。新近研究表明LncRNA在神经系统损伤中发挥着重要作用。本课题组首次利用LncRNA测序发现，LncRNA MIAT(MIAT)高表达于DHLF后的大脑皮层，文献报道MIAT广泛参与凋亡调控，而神经元凋亡是DHLF后脑损伤的主要表现，提示MIAT可能参与DHLF后脑缺血再灌注损伤。通过生物信息学分析及预实验发现Cryab可能为MIAT的关键靶点，有研究显示Cryab在抗凋亡中起重要作用。据此推测：LncRNA MIAT通过调控Cryab基因参与DHLF后脑缺血再灌注损伤。课题组拟建立小鼠DHLF细胞及动物模型，采用过表达与沉默策略，结合RIP和RNA拉下实验，验证MIAT和Cryab相互调控关系，阐明其在DHLF后脑缺血再灌注损伤中的作用，为开发DHLF脑保护策略提供新的理论依据。

深低温低流量（DHLF）后脑缺血再灌注损伤是复杂先心病体外循环术后亟待解决的问题，lncRNA调控基因表达已经成为一种新的生物学研究模式。本项目首次利用lncRNA测序技术，发现lncRNA MIAT高表达于DHLF后细胞、小鼠神经元中，此外在临床DHLF患儿血浆中也发现其高表达，因此我们推测lncRNA MIAT参与DHLF后脑缺血再灌注损伤。我们采用siRNA和慢病毒质粒构建沉默和过表达lncRNA MIAT细胞和小鼠，构建DHLF后流式细胞仪、WB、水迷宫等检查观察lncRNA MIAT对神经元凋亡的影响。发现过表达lncRNA MIAT后可以显著减轻DHLF后脑缺血再灌注损伤。随后我们通过生物信息学分析、荧光素酶报告基因和RNA免疫共沉淀技术分析验证lncRNA MIAT、miR-22和RCryab之间的靶向关系，结果证实lncRNA MIAT可能是通过竞争性结合miR-22来调控Cryab的表达，改善神经元的凋亡。这一研究结果为DHLF后脑缺血再灌注损伤的治疗提供了新的策略。