lncRNA调控人类心肌细胞增殖能力转变过程的研究

It has been found that the heart loses its regenerative ability which exists in the fetal heat when the cardiomyocyte proliferation declines in the adult heart, which restricts the therapy effects of heart diseases. Although proliferation level of mammalian cardiomyocyte is high during fetal period, it starts to decrease in postnatal stage. But till now the critical factors and the molecular mechanism of which was elusive. Recently, it has become evident that LncRNAs can impact gene expression at multiple levels, and may in fact play major roles in most aspects of gene regulation, especially in the epigenetic processes that underpin cell proliferation and organogenesis. It is requisite to identify the LncRNA profile in human cardiomyocyte proliferation, and their mechanism of action requires further investigation. High-throughput microarray LncRNA screening was utilized to identify differentially expressed LncRNAs between human fetal hearts and adult hearts. The results showed that lncRNA ENST00000435695 and lncRNA KRMP1 in the fetus human heart is upregulated comparing with the adult heart. And the targets of the above two lncRNAs as CDK6 and KIF20B signal pathway were also changed significantly. Using human cardiomyocytes and transgenic mice neonatal heart as models, our research will focus on the effects of lncRNA ENST00000435695 and lncRNA KRMP1 during the transient alteration of proliferation in cardiomyocytes and disclose the key pathways controlling this proliferation. Eventually, critical lncRNAs that control cardiomyocytes proliferation will be selected and propose some new insights to cardiomyocytes repair and substitution.

哺乳动物在胚胎期的心肌细胞具有增殖能力，并能修复受损组织，但这种能力很快消失，心肌细胞增殖能力的丧失严重制约了心脏疾病的治疗。调控这一过程的关键因子和分子机制尚不明确。基于lncRNA多靶点调控的特点及其在细胞增殖和器官形成中的重要作用，推测其在心肌细胞增殖能力丧失的过程中起重要作用。前期我们的芯片结果提示，人类胚胎期心肌组织中的lncRNA ENST00000435695及KRMP1比成熟心肌的表达水平明显改变，而与lncRNA存在靶向关系、且与细胞增殖密切相关的CDK6及KIF20B的表达也发生显著了变化。本课题拟以人心肌细胞和转基因小鼠心脏为模型，研究lncRNA在心肌细胞增殖能力转变过程中的调控作用，探索其作用的关键通路，进而筛选调控心肌细胞增殖的关键lncRNA，为心肌细胞的损伤修复和替代治疗提供新思路。

成年人类的心肌细胞为终末分化细胞，在出生后早期逐渐丧失增殖能力，导致一系列的心脏疾病难以得到有效治疗。深入研究心肌细胞丧失增殖能力的发生机制，对于寻找调控心肌细胞增殖功能的关键因素，实现受损心肌的自我修复有着重要意义。lncRNA 能够以多种形式参与基因表达调控。本研究的目的是寻找在心肌细胞增殖能力改变过程中发挥关键作用 lncRNA，为心脏疾病的再生治疗提供新的靶点。我们通过基因芯片筛选在胎儿（13-17周）和成年人（30-40岁）心脏中差异表达的长链非编码RNA（lncRNA），共发现2606条差异表达的lncRNA和3079条差异表达的mRNA。通过富集分析，发现其主要分布在细胞周期通路上；经生物信息学分析，发现8条可能参与心肌细胞增殖的lncRNA。再根据同源性、心脏细胞表达差异性等特征筛选了NR_045363，对其功能进行验证。.首先，我们运用小鼠心尖切除模型来检测NR_045363表达量，发现在心肌细胞增殖高峰期，NR_045363表达量明显升高。随后，我们构建了病毒转染过表达动物模型，发现高表达NR_045363能改善小鼠心梗后心功能，同时能减少心脏缺血区纤维化面积，并促进心梗周边区心肌细胞增殖能力。其次，在细胞实验中我们用smart silencer技术建立了NR_045363敲低的胎鼠心肌细胞模型，发现胚胎心肌细胞在外源性敲低NR_045363后，增殖能力明显下降；而在NR_045363过表达腺病毒新生小鼠心肌细胞模型，其心肌细胞增殖能力相应升高。通过数据库检索和双荧光素酶基因报告系统发现NR_045363可能与miR-216a-5p互为靶点。并用挽救实验证实NR_045363能通过竞争性抑制miR-216a-5p间接调控JAK2-STAT3通路，从而控制心肌细胞增殖。.在进一步研究中，我们发现NR_045363在小鼠心梗后早期显著升高；通过干扰RNA敲低原代心肌细胞NR_045363后，通过流式和免疫荧光结果提示，大量心肌细胞发生凋亡。在体动物结果提示，过表达NR_045363能减少梗死区心肌细胞的凋亡，进而改善心梗后心功能。通过测序和生物信息学方法，发现NR_045363能激活凋亡相关p53通路。本研究的结果发表SCI文章5篇，总影响因子达到13分，参会3次。