斑马鱼miR-30e对血细胞生成的作用机制研究

doi:10.16535/j.cnki.dlhyxb.2019-104

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大连海洋大学学报 ›› 2021, Vol. 36 ›› Issue (2) : 229-233. DOI: 10.16535/j.cnki.dlhyxb.2019-104

裴田瑛1，颜帅帅1，许强华1，2，3，4*

作者信息 +

Functional mechanism of miR-30e on hematopoiesis in zebrafish

PEI Tianying1, YAN Shuaishuai1, XU Qianghua1,2,3,4*

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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。

Abstract

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.

导出引用

裴田瑛, 颜帅帅, 许强华, . 斑马鱼miR-30e对血细胞生成的作用机制研究[J]. 大连海洋大学学报, 2021, 36(2): 229-233 https://doi.org/10.16535/j.cnki.dlhyxb.2019-104

PEI Tianying, YAN Shuaishuai, XU Qianghua, . Functional mechanism of miR-30e on hematopoiesis in zebrafish[J]. Journal of Dalian Fisheries University, 2021, 36(2): 229-233 https://doi.org/10.16535/j.cnki.dlhyxb.2019-104

中图分类号： Q 71 S 917.4