转录因子TCF25调控心肌肥大的分子机制研究

By analysis on the hypertrophic cardiomyocyte model, the cardiac hypertrophic mouse model, and the human pathological cardiac hypertrophic samples, The applicants have obtained the primary result that TCF25 maybe was a repressor on cardiac hypertrophy. So far, there is no report about the functions of TCF25 in heart. The applicats identified FHL2, another cardiac hypertrophic inhibitor (and it could facilitate the acetylation of β-Cantenin), as an interaction partner of TCF25. In addition to, we have provided data that TCF25 was a trascriptional repressor might by recruiting a HDAC，which was indicated to interact with TCF4. Based on these, the applicant suggested the hypothesis that TCF25 may be the common regulator between FHL2 and HDAC (it might participate in the FHL2's function facilitating acetylation, and in the deacetlylating role of HDAC), to regulate the cardiac hypertrophic response by Wnt/β-Cantenin signaling. In this project, by using PT-PCR, qPT-PCR, Western blot, Pull-down, Co-IP, reporter gene analysis, histological and cytological analysis，cellular immunofluorescence analysis, and ChIP, combining the strategy of gene overexpression and gene silencing, the function of TCF25 suppressing cardiac hypertrophy will be studied in details. Further more, it will be studies whether TCF25 is a common regulator of FHL2 and the HDAC to regulate the acetylation level of the transcription factors of canonical Wnt signaling. It will be tried to clarify the mechanism on TCF25 to repress cardiac hypertrophy by Wnt/β-Cantenin signaling pathway in this project.

申请人等用细胞和小鼠心肌肥大模型及人心肌肥大患者样本的初步分析结果表明，TCF25可能是心肌肥大发生的抑制因子。目前尚无TCF25心脏功能的报道。同时，申请人证明TCF25与心肌肥大抑制因子FHL2（促进β-Cantenin的乙酰化）相互作用；其也可能通过招募与TCF4结合的HDAC发挥转录抑制作用。申请人据此假设TCF25可能是FHL2与HDAC两者间的共同调节因子（参与FHL2的促乙酰化作用以及HDAC的去乙酰化作用），通过经典Wnt信号调控心肌肥大的发生。本项目拟以原代大鼠心肌细胞、TCF25转基因和基因敲除小鼠为模型，通过RT-PCR、qRT-PCR、免疫印记、Pull-down、Co-IP、报告基因分析、组织学和细胞免疫荧光分析、ChIP等多种方法，结合基因过表达和基因沉默策略，研究和证实该假说，阐明TCF25通过经典Wnt信号抑制心肌肥大的分子机制。

TCF25是本科实验室首次发现的一种转录抑制因子。该基因在胚胎和成体心脏中的高表达，提示其可能在心脏发育和成体心脏功能中有重要的作用。为了探究这种转录抑制因子在成体心脏中的功能，本课题培育了TCF25转基因和基因敲除小鼠，通过RT-PCR、qRT-PCR、免疫印迹、Pull-down、Co-IP、亚细胞定位、报告基因分析、EMSA、ChIP、组织学分析等多种方法进行了研究。结果显示：1）该基因在心肌肥大患者的心肌组织以及受病理性肥大因子刺激的大鼠原代心肌细胞、小鼠心脏中的表达水平均显著降低；2）TCF25在体内调控心肌的病理性肥大反应；3）TCF25与β-Cantenin、LEF1形成三体复合物，并可直接结合在靶基因启动子上抑制经典Wnt信号通路的转录激活因子β-Cantenin和LEF1的活性，从而发挥抑制心肌肥大的效应； 4）TCF25可特异性地与HDACs的一个成员结合，抑制辅助转录因子MEF2与其靶基因启动子的结合，从而起抑制心肌肥大的作用；5）TCF25还可与心肌肥大调节因子FHL2发生相互作用，但我们的研究结果未证实这种相互作用能协同抑制心肌肥大。本项目的研究成果揭示了TCF25在心肌肥大中的作用并丰富了对心肌肥大分子机制的理解。本项目已发表标注此项目的论文15篇（其中SCI论文10篇）；培养毕业硕士研究生7名，参加国内学术交流10人次。