斑马鱼miR-30e对血细胞生成的作用机制研究  

Functional mechanism of miR-30e on hematopoiesis in zebrafish

作　　者：裴田瑛 颜帅帅 许强华[1,2,3,4] PEI Tianying;YAN Shuaishuai;XU Qianghua(College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Sustainable Exploitation of Ocean Fisheries Resources, Ministry of Education, Shanghai 201306, China;National Distant-water Fisheries Engineering Research Center, Shanghai 201306, China;Collaborative Innovation Center for Distant-water Fisheries, Shanghai 201306, China)

机构地区：[1]上海海洋大学海洋科学学院,上海201306 [2]大洋渔业资源可持续开发教育部重点实验室,上海201306 [3]国家远洋渔业工程技术研究中心,上海201306 [4]远洋渔业协同创新中心,上海201306

出　　处：《大连海洋大学学报》2021年第2期229-233,共5页Journal of Dalian Ocean University

基　　金：国家自然科学基金面上项目(31772826,31572598);国家重点研发计划项目(2018YFC0310600)。

摘　　要：microRNA(简写为miRNA)是一类内源基因编码的单链RNA分子,参与转录后基因的表达调控,在血液生成过程中起着重要作用。为研究miR-30e对鱼类血液生成的作用机制,以斑马鱼Danio rerio作为模式生物,针对青藏高原裂腹鱼血液组织中高表达的miR-30e,利用生物信息软件预测斑马鱼miR-30e的靶基因,构建含斑马鱼ALAS2-3′UTR序列的双荧光素酶报告质粒与斑马鱼miR-30e进行共转染,检测细胞双荧光素酶活性的变化;同时构建含斑马鱼ALAS2-3′UTR序列的绿色荧光蛋白质粒,与miR-30e共同显微注射斑马鱼胚胎,观察GFP荧光强度与蛋白表达量的变化。结果表明:斑马鱼胚胎在注射miR-30e模拟体48 h后,其血红蛋白表达明显减少;转染miR-30e的293T细胞双荧光素酶活性显著降低(P<0.05);斑马鱼胚胎注射miR-30e模拟体24 h后,其GFP荧光强度明显降低,且GFP蛋白表达量明显减少。研究表明,ALAS2基因是miR-30e的一个靶基因,miR-30e通过抑制ALAS2表达而抑制血红细胞的生成。MicroRNA(miRNA)as a type of single-stranded RNA molecule encoded by endogenous genes is involved in the regulation of post-transcriptional gene expression and plays an important role in hematopoiesis.In order to study the mechanism of miR-30e on hematopoiesis in zebrafish Danio rerio,the target gene of zebrafish miR-30e was predicted by bioinformatic software,based on the significantly highly expressed miR-30e in the blood tissue of the Qinghai-Tibetan plateau schizothoracine fish,and a double fluorescence plasmid containing zebrafish ALAS2-3′UTR sequence was constructed.The reporter plasmid was co-transfected with zebrafish miR-30e,and cell luciferase activity was subsequently examined.Meanwhile,green fluorescent protein particles containing zebrafish ALAS2-3′UTR sequence were established,co-micro-injected with miR-30e into zebrafish embryos,and the GFP fluorescence intensity and protein expression changes were subsequently observed.The results showed that the expression of hemoglobin was significantly reduced in the zebrafish embryos micro-injected with miR-30e mimics 48 hours after micro-injection,the relative activity of luciferase in the 293T cells transfected with miR-30e was significantly reduced(P<0.05).Moreover,the GFP fluorescence intensity and the GFP protein expression in the zebrafish embryos micro-injected with miR-30e mimics 24 hours after micro-injection were significantly reduced.It is concluded that ALAS2 is one of the target genes of miR-30e which inhibits erythropoiesis by reducing the expression levels of ALAS2.

关 键 词：斑马鱼 miR-30e ALAS2 血红细胞生成 微小RNA 

分 类 号：Q71[生物学—分子生物学] S917.4[农业科学—水产科学]