环状RNA_circTBCD/miR-138/MLK3信号轴在心肌慢性缺氧适应中的作用研究

Compensatory adaptive responses could be evoked when cardiomyocytes are exposed to chronic hypoxia. However, its regulatory mechanism remains unclear. Based on our previous research, miR-138 was found to be up-regulated in hypoxic cardiomyocytes, which could promote cell survival in chronically hypoxic conditions via inhibiting MLK3 expression. In preliminary experiments, myocardial samples of patients with congenital heart disease were collected to carry on circular RNA chip detection. We found that circTBCD was significantly decreased in hypoxic cardiomyocytes. By using bioinformatic analysis, circTBCD was found to directly bind with miR-138. In addition, down-regulation of circTBCD could alleviate myocardial apoptosis induced by hypoxia. Therefore, we speculate that chronic hypoxia could induce down-regulation of circTBCD. Because of competing endogenous RNA effects between circTBCD and miR-138, the inhibitory role of miR-138 on its target MLK3 could be enhanced. The activation of its downstream signaling pathway is subsequently blocked, thus attenuating myocardial apoptosis and pathological remodeling. The present research intends to establish chronically hypoxic cell and animal models, and interfere the expression of circTBCD as well as miR-138 to explore their effects on hypoxic myocardiocytes. We aim to clarify how circTBCD regulates miR-138, and elucidate the specific mechanism that circTBCD / miR-138 / MLK3 signaling pathway mediates myocardial adaptation to chronic hypoxia, thereby providing theoretical evidence and potential targets for clinical cardioprotection.

心肌经历慢性缺氧时会出现代偿性适应，但是其调控机制有待研究。既往研究发现，miR-138在缺氧心肌中表达增高，通过靶向抑制MLK3可促进心肌存活。在前期实验中通过采集先心病患者心肌标本进行环状RNA芯片检测，我们发现circTBCD在缺氧心肌中表达显著降低，生物信息学软件提示其与miR-138存在直接结合位点，并且下调circTBCD表达后能抑制心肌凋亡。因此推测：缺氧诱导circTBCD表达下调，由于与miR-138存在ceRNA作用，进而增强miR-138对靶蛋白MLK3的调控，抑制其表达及下游通路激活，从而减轻心肌凋亡及病理性重构。本研究拟干扰circTBCD及miR-138表达以研究circTBCD在缺氧心肌中的作用及机制，明确circTBCD对miR-138的直接调控，证实circTBCD/miR-138/MLK3信号轴参与心肌慢性缺氧适应，为临床心肌保护提供有效的靶点。

在心肌遭受慢性缺氧刺激时会启动心肌代偿性适应保护过程，但是其调控机制有待研究，探明心肌慢性缺氧适应的有效靶点并进行干预治疗，同时寻找心肌缺氧损伤的早期预测标志物，是临床上的重要难题。本研究在前期工作基础上，通过对慢性缺氧心肌差异化表达基因的生物信息学分析，筛选出相关热点基因及通路。发现circTBCD在紫绀型先心病患者心肌中表达显著降低，建立慢性缺氧细胞模型，证实circTBCD的低表达能够减轻缺氧所致的心肌凋亡，并且干扰circTBCD后miR-138的表达显著升高。纳入在我科行心脏手术的成年患者，采集右室流出道心肌标本，发现miR-138表达与体外循环术后心肌损伤密切相关。c-Jun是miR-138调控的下游转录因子，慢性缺氧条件下心肌细胞中c-Jun的转录、蛋白表达及其磷酸化水平出现上调。c-Jun的敲减使缺氧心肌细胞的活力显著下降，增加了PTEN磷酸化水平以及减少了Akt磷酸化水平，并且抑制了细胞周期从G1期进展到S期和G2期。金丝桃苷（Hyperoside，Hyp）是近年来研究较多的一种中草药提取物，我们发现心肌细胞中加入Hyp后能显著增加miR-138的表达。在缺氧条件下，Hyp可以通过减轻凋亡及氧化应激反应进而起到心肌保护的作用，当同时加入miR-138拮抗剂后该作用会被抵消。通过对本中心先心病患儿进行回顾性研究我们发现，中性粒细胞淋巴细胞比率（neutrophil lymphocyte ratio，NLR）、血小板淋巴细胞比值（Platelet lymphocyte ratio，PLR）及红细胞体积分布宽度（Red blood Cell distribution width，RDW）与术后早期临床结局及多器官功能损伤具有一定的相关性，并能够预测术后机械通气时间延长的发生。通过本项目的进行，已初步证实了circTBCD/miR-138/MLK3/c-jun信号轴是参与心肌慢性缺氧适应的关键通路，而金丝桃苷通过干预该通路能减轻缺氧所致心肌损伤，可作为临床心肌保护的潜在治疗措施，并且发现了部分血清学标志物能够预测心脏术后早期临床结局。