RNA甲基化转移酶NSUN5缺失通过调控OCT4致先天性心脏病机制研究

英文摘要：The pathogenesis of congenital heart disease (CHD) is complicated. It affects about 28% of the birth defect population. Recently, study showed that the methylation level of RNA affected the process of embryogenesis, that even led to many diseases. A human embryonic stem cells line with NSUN5/Knock-down (hESCs-NSUN5KD) that was established in our lab, research results showed that NSUN5KD decreased the level of m5C-RNA, and inhibited hESCs-NSUN5KD differentiation into cardiac precursor cells by down-regulating OCT4. NSUN5 knock-out (NSUN5KO) mouse model was obtained from our laboratory, pre-experiment results revealed that NSUN5KO reduced the expression level of m5C-RNA and inhibited cardiogenesis via down-regulating OCT4. Our aim is to: 1. Molecular biology and cell biology techniques will be used to assess the mechanism that whether NSUN5KD disturb the m5C methylation level in hESCs and inhibit the process of hESCs differentiation into cardiac cells by down-regulating OCT4. 2. According to the mouse model with NSUN5KO, molecular biology and cell biology techniques will be employed to assess the mechanism that whether NSUN5KO decreased the m5C methylation level in mouse model and inhibit the process of cardiogenesis via down-regulating OCT4. Our research purpose is figure out that the mechanism between NSUN5-OCT4, m5C-RNA level and CHD. Our study will provide theoretical and scientific basis to CHD clinical therapy and pathogenic factors in NSUN5-OCT4 signal pathway.

先天性心脏病（CHD）发病原因复杂，占出生缺陷28%。研究显示RNA甲基化水平改变可影响胚胎发育过程，并导致多种疾病发生。课题组构建RNA甲基化转移酶NSUN5/Knock-down人胚胎干细胞系（hESCs-NSUN5KD），发现NSUN5KD导致m5C-RNA甲基化水平升高，并通过下调OCT4抑制hESCs分化为心肌前体细胞。构建NSUN5敲除小鼠模型，发现NSUN5KO下调OCT4表达并抑制小鼠心脏发生。本课题拟在成功构建细胞和动物模型基础上开展进一步机制研究：①应用细胞模型，分析NSUN5KD对m5C-RNA甲基化水平和通过调控OCT4对hESCs分化至心肌细胞阶段的影响及机制。②应用小鼠模型，分析NSUN5KO对心脏m5C-RNA甲基化水平及过调控OCT4对小鼠心脏发生过程的影响及机制。本研究阐明NSUN5-OCT4在心脏发生中的机制作用，为CHD发病机理分析及临床诊治奠定基础。

先天性心脏病（congenital heart disease, CHD）是由于心脏或者胸腔内大血管在胚胎发育过程中发生障碍所致的先天性结构或功能异常。约占出生缺陷的28%，是造成婴幼儿死因或残疾的主要原因之一。影响心脏发育并与CHD发生相关的核心转录因子有NKX2.5、GATA4和TBX5，而目前有报道指出GATA4基因CpG岛区域过甲基化修饰可促进CHD发生。另有研究提示，TBX5启动子区域甲基化水平与CHD发生具有直接关联性；同时临床案例数据统计指出高甲基化水平与复杂性CHD具有相关性，而低甲基化水平与唐氏综合征引发CHD相关。m5C-RNA甲基化调控方式直接与多种疾病相互关联。研究显示，m5C广泛存在于编码及非编码RNA中，通过对RNA加工、RNA稳定性、RNA降解的影响调控着mRNA翻译程度和寿命，并通过以上方式引发细胞凋亡，应激反应异常，生物节律改变和影响代谢过程。m5C-RNA甲基化修饰与DNA和组蛋白修饰方式相似是一种动态过程。本研究阐述了NSUN5在心脏流出道发育过程中发挥的作用，首次通过流行病学方法系统鉴定到NSUN5功能性突变在法洛四联症患者中富集，生物功能学实验证实Nsun5缺失能够引起法洛四联症相关的流出道发育迟缓，其通过调节修饰水平影响下游基因Tpm1的翻译，Tpm1蛋白表达下降影响细胞生长通路中相关基因表达从而导致小鼠心脏组织细胞增殖减缓所实现，提示Nsun5/Tpm1信号轴在心脏流出道发育中的重要性。本研究结果阐述了m5C甲基化酶NSUN5调控m5C修饰在心脏发育中的作用机制和方式，为法洛四联症遗传学病因探索提供了新的视角。