摘 要：microRNAs（miRNAs）是一类广泛参与各种生理过程的小非编码RNA，它们通过与靶标mRNA结合来调控蛋白质编码基因的表达。miR-194基因在肝脏组织中高表达，是人类早期肝损伤诊断的关键血清生物标志物。然而，该基因在肝损伤进展中的作用尚不清楚。生物信息学分析结果表明，miR-194b基因在多个物种中高度保守。为了揭示该基因在肝脏发育中的作用，本研究利用CRISPR/Cas9基因编辑技术构建miR-194b基因敲除的斑马鱼品系，并对其在肝脏发育中的作用进行初步探讨。首先利用在线网站设计两个miR-194b的特异性靶点，通过PCR扩增获得sgRNA模板DNA，DNA转录得到sgRNA。将sgRNA和Cas9蛋白共注射到斑马鱼1细胞胚胎中。将注射胚胎养至2月龄后筛选得到F0代嵌合突变体。F0代个体与野生型斑马鱼杂交得到F1代杂合突变体，经基因型鉴定及Sanger测序筛选出相同突变类型的F1代个体，F1代杂合子自交最终得到F2代纯合突变体。miR-194b纯合突变体未表现出明显的发育异常且具备正常的繁殖能力。miR-194b过表达试验结果表明，斑马鱼胚胎呈剂量依赖性发育畸形，其畸形的主要表现为肌节紊乱、心包水肿、肝脏变小、鱼鳔缺失，这证明miR-194b在斑马鱼胚胎早期发育和器官生成中发挥了重要作用。本研究成功构建了miR-194b敲除斑马鱼，为未来探究miR-194在肝脏发育中的作用提供了有效模型。
关键词：斑马鱼；miR-194b基因；肝脏发育；microRNAs；CRISPR/Cas9
中图分类号：Q812                        文献标志码：A                    DOI：10.3969/j.issn.1007-7146.2025.04.005

Abstract: microRNAs (miRNAs), a type of small non-coding RNA widely involved in various physiological processes, regulates the expression of protein-coding genes by binding to target mRNA. The miR-194 gene is highly expressed in the liver and serves as a key serum biomarker for diagnosing early liver injury in humans. However, the role of miR-194 in the progression of liver injury remains unclear. The results of bioinformatics analysis indicated that the miR-194 gene was highly conserved in various species. To reveal the role of miR-194 gene in liver development, this study utilized CRISPR/Cas9 gene editing technology to construct miR-194b knockout lines, and to preliminarily investigate the role of miR-194b gene in liver development. First, two target sites of miR-194b gene were designed using an online website. The sgRNA template DNA was obtained by PCR amplification, and then DNA was transcribed to generate sgRNA. The sgRNA and Cas9 protein were co-injected into the zebrafish 1-cell embryos. After injection, the embryos were raised to 2 months of age and screened to obtain the F0 generation chimeric mutants. F0 chimeras were crossed with wild-type zebrafish to obtain F1 heterozygotes, which were subsequently genotyped and screened for the same mutation type by Sanger sequencing. F1 generation heterozygotes were crossed and genotyped to obtain F2 generation of homozygous mutants. The miR-194b homozygous mutants exhibited no obvious phenotypic abnormalities and were fertile. Overexpression of miR-194b caused does-dependent developmental malformations, including disorganized myotomes, pericardial edema, smaller liver, and absence of swim bladder, demonstrating that the miR-194b plays an important role in the early development and organogenesis of zebrafish embryos. In this study, we successfully constructed a zebrafish miR-194b knockout line, which lays the foundation for an in-depth study of the role of miR-194 in liver development.
Key words: zebrafish; miR-194b gene; liver development; microRNAs; CRISPR/Cas9
（Acta Laser Biology Sinica, 2025, 34(4): 319-327）