| 摘要: |
| microRNA为短链非编码RNA，通过与靶基因3′UTR序列互补在转录后水平发挥作用。已有研究表明，microRNA在红细胞发生过程中起着重要的调控作用。南极冰鱼是目前已知的唯一仅具有无功能性血红细胞的脊椎动物。前期研究提示，独角雪冰鱼（Chionodraco hamatus）头肾中高表达的microRNAs可能抑制着冰鱼红血球的发生。本研究针对南极冰鱼头肾中高表达的miR-7132，运用斑马鱼显微注射、双荧光素酶报告系统，并结合靶基因预测等手段研究了miR-7132对血红细胞发生的作用机制。结果表明：斑马鱼胚胎注射miR-7132后，固蓝染色显示斑马鱼胚胎红细胞中血红蛋白的表达显著下降，这表明miR-7132的过表达抑制了血红细胞生成。通过转录组数据结合比对冰鱼的全基因组序列，获得了独角雪冰鱼血红细胞生成的血红素生物合成的限速酶（5′-aminolevulinate synthase 2，ALAS2）基因3′UTR序列。构建含ALAS2基因3′UTR序列的双荧光素酶报告质粒，并与miR-7132共转染293T细胞检测荧光素酶活性变化；构建包含绿色荧光蛋白（green fluorescent protein，GFP）的表达质粒与miR-7132共同注射斑马鱼胚胎，检测GFP荧光强度与蛋白表达量变化。结果显示，转染miR-7132的293T细胞荧光素酶相对活性显著降低，ALAS2基因是miR-7132的一个靶基因。斑马鱼体内注射miR-7132的GFP荧光强度显著降低，且GFP蛋白表达量显著减少。本研究揭示了miR-7132对血红细胞生成的抑制作用，miR-7132通过抑制ALAS2基因的表达而抑制南极冰鱼血红细胞的发生。 |
| 关键词: 南极冰鱼 miR-7132 ALAS2 红细胞发生 |
| DOI：10.11693/hyhz20151200311 |
| 分类号: |
| 基金项目:国家自然科学基金面上项目，31572598号；国家自然科学基金重大研究计划培育项目，91131006号；上海市教育发展基金会和上海市教育委员会曙光计划，13SG51号；教育部科学技术研究项目，213013A号；上海市教委水产学高峰学科项目。 |
附件 |
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| ERYTHROPOIESIS STUDY OF MIR-7132 IN ANTARTCTIC ICEFISH CHIONODRACO HAMATUS |
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HU Xing-Xing1, WANG Cong-Cong1,2, CHAN Jiu-Lin1, XU Qiang-Hua1,2,3,4
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1.College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China;2.Key Laboratory of Sustainable Exploitation of Ocean Fisheries Resources, Ministry of Education, Shanghai 201306, China;3.National Distant-water Fisheries Engineering Research Center, Shanghai Ocean University, Shanghai 201306, China;4.Collaborative Innovation Center for Distant-water Fisheries, Shanghai 201306, China
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| Abstract: |
| microRNAs are small non-coding RNAs acting at post-transcriptional level by binding the target gene 3′UTR (untranslated region), and have an important effect on the erythropoiesis. Chionodraco hamatus is the only known vertebrates with a few non-functional erythrocytes. Previous findings showed that the highly upregulated expression of microRNAs in the head kidney of the animal could inhibit its erythropoiesis. By applying zebrafish microinjection, dual-luciferase reporter, and the target prediction method, we studied the erythropoiesis of miR-7132 that highly expressed in the head kidney. Results show that, after being injected with miR-7132, the expression level of hemoglobin in zebrafish embryos dramatically reduced as observed after o-dianisidine staining, indicating that the overexpression of miR-7132 repressed the erythropoiesis process in zebrafish embryos. The 3′UTR of ALAS2 (i.e., 5′-aminolevulinate synthase 2), the limited enzymes of hemoglobin production, was obtained from transcriptome sequencing data blasting unpublished icefish genome sequences. The dual-luciferase vector containing the 3′UTR of ALAS2 was constructed and co-transfected with miR-7132, and the activity of luciferase was detected. The expression vector containing the GFP (green fluorescent protein) fluorescent protein was constructed. Zebrafish embryos were then micro-injected with the mixture of miR-7132 and GFP plasmid, and the expression of GFP protein was detected by the western blotting. Therefore, miR-7132 could reduce significantly the relative activity of luciferase in 293T cells. The GFP fluorescent intensity could be decreased significantly when injected with miR-7132; and miR-7132 could regulate erythropoiesis in the Antarctic icefish by repressing the expression of ALAS2. |
| Key words: Antarctic icefish miR-7132 ALAS2 erythropoiesis |
