低温敏感的微小RNA-7参与调节深低温低流量下Akt信号通路的分子机制

The expression and transcription of microRNAs has been demonstrated to play an impotant role of regulation the cell biological behaviour.MicroRNA expression changing of biological phenomena caused by DHLF brought our attention.It has been reported there were close relationship of miR-7 expression and Akt signaling pathways' regulation. MicroRNA expression and its molecular mechanism interaction with Akt signaling pathways is not clear in the DHLF in brain damage during surgery of congenital heart disease(CHD).Our previous study shown there were active expression of miR-7 in DHLF brain tissue.In this study we hypothesis there would be the difference of phase of AKT signaling pathway and dowstream apoptosis molecular expression. DHLF rats and S5Y5 cells were be used as model, analysis neural tissue pathological changes and organizational positioning,determine miR-7 with AKT signaling pathways potential interaction sites in DHLF rats, find the mutual relationship for both and the effects of cells in low temperature low oxygen state. This research aim to reveal temperature-sensitive miRNA and Akt signaling pathways in DHLF brain protection, it has important significance to understand molecular mechanism in DHLF, moreover, it also can provide the theory basis to find the new treatment stratege and judge prognosis.

MicroRNA的表达可在基因转录后水平对细胞的生物学行为起重要的调控作用。而低温引起microRNA表达改变的生物学现象引起了我们的关注，研究表明miR-7表达与Akt信号通路的调控作用密切相关，而在先心病深低温低流量(DHLF)相关脑损伤中miRNA的表达与Akt信号传导通路相互作用及分子机制尚不清楚。我们前期研究表明深低温低流量下脑组织中mir-7的表达活跃。本课题拟以DHLF大鼠及S5Y5细胞为模型，分析经DHLF后神经组织病理学改变，AKT信号通路及下游凋亡途径分子表达特点，miR-7参与调节Akt信号通路潜在的关键位点，明确相互作用关系及对低温低氧状态下细胞的作用。本研究对揭示miRNA与Akt信号通路在深低温低流量脑保护作用的分子机制及潜在药物干预靶点提供理论依据。

深低温低流量（DHLF）后脑缺血再灌注损伤是先心病体外循环术后最常见并发症，miRNA是一类具有调控功能的高度保守的内源性非编码RNA，通过碱基互补配对原则选择性地同靶基因mRNA的3’-UTR相结合，在转录后水平阻遏靶mRNA的翻译。我们前期研究发现，miR-7在DHLF后脑组织中表达量明显增加，提示其可能参与了DHLF后脑缺血再灌注损伤，我们既往研究已经证实Akt信号通路在DHLF后脑保护中发挥重要作用，因此我们推测miR-7可能通过调控Akt信号通路中的关键位点从而在DHLF后的脑损伤中发挥重要作用。基于上述猜想，我们通过分离原代神经元细胞，建立miR-7过表达和抑制神经元细胞，模拟DHLF体外条件建立深低温低氧细胞模型，24h后收集细胞进行功能检查，通过流式细胞学、ELISA及WB等实验手段，证实了miR-7在小鼠原代神经元细胞深低温低氧模型中具有保护作用。通过双荧光素酶报告基因，我们发现miR-7可能与抑制PTEN蛋白表达，负性激活PI3K/Akt信号通路有关。为了在动物体内验证我们的猜想，我们通过侧脑室注射miR-7 agomir和antagomir，建立miR-7过表达和抑制小鼠，DHLF后不同时间点提取脑皮层组织。运用神经功能缺损评分、HE染色、TUNEL染色及WB等实验技术，结果证实miR-7在小鼠DHLF脑缺血再灌注损伤中具有一定的保护作用，其机制可能是通过与PTEN特异性结合并抑制其表达，随后激活PI3K/Akt信号通路，从而发挥脑保护作用。我们的研究结果为临床研究DHLF后神经系统相关并发症提供了一定的理论基础，并使miR-7成为潜在的脑保护治疗靶点，为临床防治DHLF后神经系统损伤提供新的治疗策略。