Abstract: Antarctic icefish (Chionodraco hamatus) is the only known vertebrate that lacks hemoglobin and does not have functional red blood cells. Compared with other Antarctic fish, C. hamatus has thicker blood vessels and more capillaries. Preliminary study has showed that miR-204 was expressed at significantly lower levels in blood vessels of C. hamatus than that of Trematomus bernacchii, its relative species. It was suggested that miR-204 might play important roles in the regulation of vascular development. Aim to prove the hypothesis, this paper selected model animal zebrafish as experiment material, used molecular biology techniques including embryo micro-injection, target gene prediction and dual-luciferase reporter assay system, investigated the roles of miR-204 in vascular development. The results showed that inhibiting miR-204 caused capillary hyperplasia of the trunk blood vessels in C. hamatus embryos. The luciferase assay showed that miR-204 significantly repressed the transcriptional activity of its target gene trib3. Meanwhile, after injecting miR-204 and trib3 together into zebrafish embryos, the GFP fluorescence intensity and GFP protein quantity were significantly reduced, indicating that miR-204 had an inhibitory effect on trib3. Trib3 was demonstrated initially to be a pseudokinase that inhibited mitosis in Drosophila melanogaster embryos and germ cells, and subsequently found to participate the regulation of atherosclerosis and diabetic angiofibrosis. Therefore, we proposed that miR-204 possibly regulated vascular development by repressing gene expression of trib3.

Key words: Antarctic icefish, miR-204, trib3, vascular development

摘要： 南极独角雪冰鱼（Chionodraco hamatus）是目前已知的唯一缺乏血红蛋白、且不具有功能性血红细胞的脊椎动物。与相同生境下的其他南极鱼相比，其血管明显变粗，毛细血管增多。前期对南极独角雪冰鱼与近缘种伯氏肩孔南极鱼（Trematomus bernacchii）的血管microRNAs表达进行对比，发现南极独角雪冰鱼的miR-204的表达与伯氏肩孔南极鱼间存在显著差异，推测miR-204极有可能参与了南极冰鱼血管发育的调控。为证明此假设，运用斑马鱼（Barchydanio rerio）这一模式生物，利用斑马鱼胚胎显微注射技术、靶基因预测、双荧光素酶报告系统等一系列分子生物学手段对miR-204在斑马鱼血管发育中的调控作用进行探究。结果显示，抑制miR-204表达能使斑马鱼胚胎躯干部毛细血管增生。荧光素酶活性分析表明，miR-204可以显著降低trib3的表达，同时在斑马鱼胚胎中共注射miR-204和trib3后的GFP萤光强度显著降低，GFP蛋白量减少，说明miR-204对trib3有抑制作用。trib3最初发现是抑制果蝇胚胎和生殖细胞有丝分裂的假激酶，后续研究发现其参与动脉粥样硬化的调控以及糖尿病血管纤维化。综上，推测miR-204可能通过抑制trib3的表达进而调控毛细血管的增生。

关键词: The Role of miR-204 on Vascular Development in , Zebrafish Embryos