LncRNA GAS5作为ceRNA调控miR-23a-3p参与颅脑创伤继发性损伤的作用和机制

Secondary injury determines largely the patient outcome following traumatic brain injury (TBI) and it is of great scientific and clinical significance to search the key molecule during TBI onset and progress. Long non-coding RNA (LncRNA) plays a crucial role in the regulation of multiply physiological and pathological processes, and therefore, is one of the hot topics in current biomedical research. We previously found that there are 271 lncRNAs expressed abnormally in the rat hippocampus after acute TBI, among which the concentration of GAS5 increases significantly as many as three folds compared with the control. In addition, after GAS5 was knocked down, the number of apoptotic cells in the field of rat cortex and hippocampus reduces obviously, coupled with the increased level of miR-23a-3p. Thus, it is speculated that GAS5 has the ability of functioning as a ceRNA to bind miR-23a-3p and regulate cell apoptosis, via which to take part in the secondary injury of TBI. Three parts of experiments are designed in our research application as follows: 1. to find out the expression pattern of GAS5 and the correlation to TBI in vivo and vitro models; 2. to explicit the role of GAS5 in the cell apoptosis following TBI via gain/loss-of-function methods; 3. to discuss the relevant mechanisms of GAS5 to bind miR-23a-3p and to regulate the downstream gene, APAF-1. We hope the above research plans will provide experimental and theoretical basis for GAS5 participating in the secondary injury of TBI.

颅脑创伤（TBI）继发性损伤是决定患者预后的重要病理基础，寻求疾病发生发展中的关键分子具有重要的科研和临床意义。lncRNA在众多生理病理学过程中均有极其重要的调控作用，是当前生物医学研究领域的热点。前期研究发现TBI后大鼠海马组织内多达271条lncRNA表达失调，其中GAS5水平明显升高，为对照组3倍以上；敲低GAS5表达后海马和皮层区域的凋亡细胞数量明显减少，同时miR-23a-3p水平明显升高。因此推测GAS5可作为ceRNA结合miR-23a-3p并通过调控细胞凋亡参与TBI继发性损伤。本项目拟从三方面验证该假说：1.构建体内体外TBI模型研究GAS5表达规律及与TBI的相关性；2.过表达或敲低GAS5确定其对TBI后细胞凋亡的影响；3.探讨GAS5结合miR-23a-3p进而调控下游靶基因APAF-1的机制。从而为GAS5参与TBI继发性损伤提供实验基础和理论依据。

颅脑创伤（TBI）致死致残率居全身各类创伤之首，是医疗卫生和社会经济学的重大难题。其中，继发于创伤后的神经细胞凋亡是引起预后不良的重要病理生理机制。近年来，表观遗传学尤其长链非编码RNA（lncRNA）在TBI发病机制中的作用逐步受到研究者关注。项目申请人前期研究发现大鼠TBI后多条lncRNA表达失调，其中lncRNA GAS5表达显著升高，下调GAS5可减轻大鼠创伤脑组织的细胞凋亡，生物信息学分析推测GAS5可能通过竞争性内源RNA（ceRNA）机制参与TBI继发性损伤的调节。本项目构建了液压冲击脑损伤的动物模型和原代培养神经元牵张损伤的细胞模型，从GAS5的表达模式、体内体外功能和分子机制角度进行相关研究。结果发现：（1）GAS5在大鼠创伤皮层和伤侧海马表达明显升高，而对侧皮层与海马基本不表达，细胞类型上以神经元表达为主，且大多定位于细胞浆；（2）凋亡蛋白免疫印迹、培养液LDH测定、TUNEL染色、FJB染色以及动物神经行为学评估证实调控GAS5可减少体外体内模型的细胞凋亡和神经元变性；（3）GAS5作为ceRNA结合miR-23a-3p，使后者对其下游靶分子APAF1的抑制作用降低，APAF1蛋白表达增加，促进脑创伤后神经元凋亡和变性。综上，通过本项目研究，我们揭示了GAS5/miR-23a-3p/APAF1通路在TBI细胞凋亡中的功能与机制，深化了对非编码RNA在TBI继发性损伤中作用的认识，为进一步探明TBI病理生理学机制和开发新的治疗策略提供参考依据。