Zhu Hong, Zhang Yanfeng, Zhang Chengliang, Xie Zhongshang
Department of Cardiovascular Surgery, Xiangya Hospital, Central South University, Changsha, China.
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China.
Front Cardiovasc Med. 2021 Dec 23;8:773573. doi: 10.3389/fcvm.2021.773573. eCollection 2021.
Pathological tissue remodeling such as fibrosis is developed in various cardiac diseases. As one of cardiac activated-myofibroblast protein markers, CKAP4 may be involved in this process and the mechanisms have not been explored. We assumed that CKAP4 held a role in the regulation of cardiac fibrotic remodeling as an RNA-binding protein. Using improved RNA immunoprecipitation and sequencing (iRIP-seq), we sought to analyze the RNAs bound by CKAP4 in normal atrial muscle (IP1 group) and remodeling fibrotic atrial muscle (IP2 group) from patients with cardiac valvular disease. Quantitative PCR and Western blotting were applied to identify CKAP4 mRNA and protein expression levels in human right atrium samples. iRIP-seq was successfully performed, CKAP4-bound RNAs were characterized. By statistically analyzing the distribution of binding peaks in various regions on the reference human genome, we found that the reads of IP samples were mainly distributed in the intergenic and intron regions implying that CKAP4 is more inclined to combine non-coding RNAs. There were 913 overlapping binding peaks between the IP1 and IP2 groups. The top five binding motifs were obtained by HOMER, in which GGGAU was the binding sequence that appeared simultaneously in both IP groups. Binding peak-related gene cluster enrichment analysis demonstrated these genes were mainly involved in biological processes such as signal transduction, protein phosphorylation, axonal guidance, and cell connection. The signal pathways ranking most varied in the IP2 group compared to the IP1 group were relating to mitotic cell cycle, protein ubiquitination and nerve growth factor receptors. More impressively, peak analysis revealed the lncRNA-binding features of CKAP4 in both IP groups. Furthermore, qPCR verified CKAP4 differentially bound lncRNAs including LINC00504, FLJ22447, RP11-326N17.2, and HELLPAR in remodeling myocardial tissues when compared with normal myocardial tissues. Finally, the expression of CKAP4 is down-regulated in human remodeling fibrotic atrium. We reveal certain RNA-binding features of CKAP4 suggesting a relevant role as an unconventional RNA-binding protein in cardiac remodeling process. Deeper structural and functional analysis will be helpful to enrich the regulatory network of cardiac remodeling and to identify potential therapeutic targets.
诸如纤维化之类的病理性组织重塑在各种心脏疾病中都会发生。作为心脏激活的肌成纤维细胞蛋白标志物之一，CKAP4可能参与了这一过程，但其机制尚未得到探索。我们假设CKAP4作为一种RNA结合蛋白在心脏纤维化重塑的调控中发挥作用。利用改进的RNA免疫沉淀和测序技术（iRIP-seq），我们试图分析心脏瓣膜病患者正常心房肌（IP1组）和重塑纤维化心房肌（IP2组）中与CKAP4结合的RNA。应用定量PCR和蛋白质印迹法来鉴定人右心房样本中CKAP4的mRNA和蛋白表达水平。成功进行了iRIP-seq，对与CKAP4结合的RNA进行了表征。通过对参考人类基因组上各个区域结合峰的分布进行统计分析，我们发现IP样本的读数主要分布在基因间和内含子区域，这意味着CKAP4更倾向于结合非编码RNA。IP1组和IP2组之间有913个重叠的结合峰。通过HOMER获得了前五个结合基序，其中GGGAU是在两个IP组中同时出现的结合序列。结合峰相关基因簇富集分析表明，这些基因主要参与信号转导、蛋白质磷酸化、轴突导向和细胞连接等生物学过程。与IP1组相比，IP2组中变化最大的信号通路与有丝分裂细胞周期、蛋白质泛素化和神经生长因子受体有关。更令人印象深刻的是，峰分析揭示了两个IP组中CKAP4的lncRNA结合特征。此外，qPCR验证了与正常心肌组织相比，CKAP4在重塑心肌组织中差异结合包括LINC00504、FLJ22447、RP11-326N17.2和HELLPAR在内的lncRNA。最后，CKAP4在人类重塑纤维化心房中的表达下调。我们揭示了CKAP4的某些RNA结合特征，表明其作为一种非常规RNA结合蛋白在心脏重塑过程中发挥相关作用。更深入的结构和功能分析将有助于丰富心脏重塑的调控网络并识别潜在的治疗靶点。
