心肌缺血再灌注损伤的调控新分子——BTF

Myocardial ischemia reperfusion injury (MRI/I) is the major pathological factors of acute myocardial ischemia injury aggravated and mortality increased. The mechanism of MIR/I remained unclear and lacked effective therapeutic measures. Our study researches recently found that the expression of BTF increased in I/R myocardial in contrast to wild type mice, and BTF act as a transcription factor activating the expression of P53 and regulating apoptosis. Furthermore, we demonstrated the interaction of BTF and lncRNA-343. Increasingly, the mRNA level of lncRNA-343 decreased in MRI/I comparison with control group, and it has a significant relationship with apoptosis. Based on what we found, we hypothesis that: in contract with wild type, the expression of lncRNA-343 decreased and attenuated the interaction with BTF, free form of BTF elevated in MIR/I mice, the enhanced BTF activated the transcription of P53 and induced apoptosis. Our study will construct the mice and adopted RIP, RNA pull-down and molecular biological techniques illuminated the mechanism of lncRNA-343 and BTF, established a novel regulatory pathway, discovered a new therapeutic target and provided a systematically strategy of pathology and clinic treatment.

心肌缺血再灌注损伤（MRI/I）是导致急性心梗后期损伤加重、死亡率升高的主要病理因素，其确切分子机制仍未清楚，缺少有效干预措施。课题组近期发现：BTF分子在小鼠MRI/I心肌组织和细胞（CMs）中表达明显升高，且可通过激活促凋亡基因p53调控CMs凋亡。进一步研究发现，BTF可与lncRNA-343特异性结合；而lncRNA-343在MRI/I中表达降低，对其干预后可影响CMs凋亡。据此，本研究提出假设：MRI/I条件下，lncRNA-343表达降低，与BTF结合减少，使游离BTF增多，促进p53激活，诱导CMs凋亡，加重MRI/I损伤。本研究将构建转基因动物，采用RIP、RNA pulldown等分子生物学技术，阐明lncRNA-343通过与BTF互作调控MRI/I的分子新机制，建立调控新通路，发现MRI/I的治疗新靶点，为系统阐明MRI/I的病理机制和有效临床防治提供全新策略。

本项目通过芯片分析发现lncRNA-CIRBIL在心肌缺血再灌注的心脏中表达降低。为进一步探究lncRNA-CIRBIL对心肌缺血再灌注损伤的作用，分别构建了心脏特异性过表达和敲除lncRNA-CIRBIL的转基因小鼠，发现过表达lncRNA-CIRBIL可以减少心脏缺血再灌注损伤面积，抑制心肌细胞凋亡，而敲除lncRNA-CIRBIL加重了心肌缺血再灌注损伤。利用Pulldown联合质谱分析手段，发现lncRNA-CIRBIL与Bcl-2相关转录因子（Bcl2-associated transcription factor，Bclaf1）能够直接相互结合。野生正常心肌细胞中，lncRNA-CIRBIL主要定位在细胞质中，而Bclaf1在细胞质和细胞核中均有分布。在lncRNA-CIRBIL过表达小鼠的心肌细胞中，Bclaf1主要定位在心肌细胞质中，而lncRNA-CIRBIL敲除小鼠的心肌细胞中，Bclaf1主要聚集在细胞核。核质分离蛋白印迹实验进一步验证了Bclaf1在细胞质和细胞核中的分布特征。心肌缺血再灌注损伤时，Bclaf1表达增加，聚集在细胞核。进一步构建心肌细胞特异性过表达Bclaf1的转基因小鼠，揭示Bclaf1的高表达可加重心脏缺血再灌注损伤，促进P53介导的心肌细胞凋亡，而敲减Bclaf1可缓解心肌缺血再灌注损伤。敲减LncRNA-CIRBIL敲除小鼠的Bclaf1时，心肌缺血再灌注损伤减轻。以上结果表明，lncRNA-CIRBIL可通过与Bclaf1结合，抑制其核转位，进而减轻心肌缺血再灌注损伤。该研究结合生物信息学、药理学和分子生物学等研究方法，发现lncRNA-CIRBIL调节心肌缺血再灌注损伤的重要作用并揭示其新机制，提出lncRNA-CIRBIL是心肌保护药物新的潜在靶分子，该发现对心肌缺血再灌注损伤的临床诊断与治疗新方法、新药物的研发具有重要意义。