Chen Feng, Yin Caiyong, Dimitropoulou Christiana, Fulton David J R
Department of Forensic Medicine, Nanjing Medical UniversityNanjing, Jiangsu, China; Vascular Biology Center, Medical College of Georgia at Augusta UniversityAugusta, GA, USA.
Department of Forensic Medicine, Nanjing Medical University Nanjing, Jiangsu, China.
Front Physiol. 2016 Jul 19;7:284. doi: 10.3389/fphys.2016.00284. eCollection 2016.
Nox5 was the last member of the Nox enzyme family to be identified. Functionally distinct from the other Nox isoforms, our understanding of its physiological significance has been hampered by the absence of Nox5 in mouse and rat genomes. Nox5 is present in the genomes of other species such as the rabbit that have broad utility as models of cardiovascular disease. However, the mRNA sequence, characteristics, and functional analysis of rabbit Nox5 has not been fully defined and were the goals of the current study.
Rabbit Nox5 was amplified from rabbit tissue, cloned, and sequenced. COS-7 cells were employed for expression and functional analysis via Western blotting and measurements of superoxide. We designed and synthesized miRNAs selectively targeting rabbit Nox5. The nucleotide and amino acid sequences of rabbit Nox5 were aligned with those of putative rabbit isoforms (X1, X2, X3, and X4). A phylogenetic tree was generated based on the mRNA sequence for Nox5 from rabbit and other species.
Sequence alignment revealed that the identified rabbit Nox5 was highly conserved with the predicted sequence of rabbit Nox5. Cell based experiments reveal that rabbit Nox5 was robustly expressed and produced superoxide at rest and in a calcium and PMA-dependent manner that was susceptible to superoxide dismutase and the flavoprotein inhibitor, DPI. miRNA-1 was shown to be most effective in down-regulating the expression of rabbit Nox5. Phylogenetic analysis revealed a close relationship between rabbit and armadillo Nox5. Rabbit Nox5 was relatively closely related to human Nox5, but lies in a distinct cluster.
Our study establishes the suitability of the rabbit as a model organism to further our understanding of the role of Nox5 in cardiovascular and other diseases and provides new information on the genetic relationship of Nox5 genes in different species.
Nox5是Nox酶家族中最后一个被鉴定的成员。与其他Nox亚型在功能上不同，由于小鼠和大鼠基因组中不存在Nox5，我们对其生理意义的理解受到了阻碍。Nox5存在于其他物种的基因组中，如兔子，兔子作为心血管疾病模型具有广泛的用途。然而，兔Nox5的mRNA序列、特征和功能分析尚未完全明确，这也是本研究的目标。
从兔组织中扩增、克隆并测序兔Nox5。通过蛋白质免疫印迹法和超氧化物测量，利用COS-7细胞进行表达和功能分析。我们设计并合成了选择性靶向兔Nox5的微小RNA（miRNA）。将兔Nox5的核苷酸和氨基酸序列与推定的兔亚型（X1、X2、X3和X4）的序列进行比对。基于兔和其他物种的Nox5 mRNA序列构建系统发育树。
序列比对显示，鉴定出的兔Nox5与预测的兔Nox5序列高度保守。基于细胞的实验表明，兔Nox5在静息状态下以及以钙和佛波酯（PMA）依赖的方式强烈表达并产生超氧化物，且对超氧化物歧化酶和黄素蛋白抑制剂二苯基碘（DPI）敏感。miRNA-1被证明在下调兔Nox5表达方面最有效。系统发育分析显示兔和犰狳的Nox5之间关系密切。兔Nox5与人类Nox5相对密切相关，但位于一个不同的聚类中。
我们的研究确定了兔子作为模式生物对于进一步理解Nox5在心血管疾病和其他疾病中的作用的适用性，并提供了关于不同物种中Nox5基因遗传关系的新信息。
