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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在心脏发育过程中起着重要的调控作用。南极冰鱼因体内缺乏功能性血红细胞,其心脏出现了补偿性增生。前期的研究提示,独角雪冰鱼心脏中特异表达的microRNAs可能与冰鱼心脏的补偿性增生相关。本研究针对南极冰鱼心脏中高表达的miR-210-5p,运用斑马鱼显微注射、靶基因预测等手段研究了miR-210-5p对心脏发育的作用机制。结果表明:斑马鱼胚胎注射miR-210-5p后,出现心包膜水肿,心脏发育畸形等现象。qRT-PCR分析显示,过表达miR-210-5p的斑马鱼胚胎中,心脏发育相关的标志性基因bmp4、smad1、gata6以及tbx2b的表达水平下调。Western blotting分析发现,bmp/smad通路中的BMP2、BMP4、SMAD1、GATA6以及TBX2B的蛋白表达水平也显著下调。通过对tbx20基因3’UTR的靶基因生物信息学预测,以及对其进行GFP荧光表达分析,发现tbx20基因可能是miR-210-5p的一个靶基因。由此推测,miR-210-5p可能通过抑制tbx20基因的表达,并调控bmp/smad通路以抑制冰鱼心脏发育。
MicroRNAs are short-chain, non-coding RNAs that act at the post-transcriptional level by complementing the 3’UTR sequence of the target gene. Studies have shown that microRNA plays an important regulatory role in cardiac development. Antarctic icefish lack of functional red blood cells due to the body, the heart appeared compensatory hyperplasia. Previous studies suggest that microRNAs specifically expressed in the heart of Unicismata arundinata may be associated with compensatory hyperplasia of icefish heart. In this study, miR-210-5p was overexpressed in the heart of Antarctic icefish. The mechanism of miR-210-5p on cardiac development was studied by microinjection of zebrafish and prediction of target genes. The results showed that the injection of miR-210-5p into zebrafish embryos resulted in the occurrence of pericardial edema and cardiac malformations. qRT-PCR analysis showed that the expression of cardiac development-related markers bmp4, smad1, gata6 and tbx2b were down-regulated in zebrafish embryos overexpressing miR-210-5p. Western blotting analysis also showed that BMP2, BMP4, SMAD1, GATA6 and TBX2B protein expression in the bmp / smad pathway was significantly down-regulated. Bioinformatics prediction of 3’UTR of tbx20 gene and analysis of its GFP expression showed that tbx20 gene may be a target gene of miR-210-5p. It is speculated that miR-210-5p may inhibit the development of icefish heart by inhibiting the expression of tbx20 gene and regulating the bmp / smad pathway.