Effects Of MiR-20b Overexpression On Zebrafish Embryonic Heart Development

Congenital heart disease (CHD) is one of the most common birth defects,which is harmful to human health. The etiology and pathogenesis have been extensively studied, but the exact mechanism is unclear. The current study shows that changes in environmental factors and genetic factors are major causes of congenital heart disease, therefore, it is significantly useful to study the role of environmental factors and genetic factors in cardiac development for the prevention, diagnosis and treatment of CHD.MicroRNAs (miRNAs) are a class of recently identified non-coding RNA molecules that play an essential role in gene expression regulation at post-transcriptional levels. They are highly conserved in evolution of species. In the present study, miRNAs gradually become hot topics in medical research, and it has been proved that miRNAs are involved in many important physiological and pathological processes, especially in the organogenesis. miR-20b is one of our screening miRNAs which were significantly up-regulated in the human heart tissues of abortive embryos due to ventricular septal defects, and none of the study of its role in embryonic heart development and the underlying mechanisms embryonic cardiac malformations study has yet reported in the papers.In this study, we used the zebrafish, which has unique advantages in the study of the cardiovascular development as a model animal to reveal the effcts of miR-20b overexpression on the early heart development in vertebrates. Firstly, we microinjected the miR-20b mimic in the single-cell stage to achieve the overexpression, and the morphological observations showed that overexpression of miR-20b in zebrafish embryos will lead to significant pericardial edema in a dose-dependent manner. The cardiac histological sections of the120hours post-fertilization (120hpf) embryos displayed that the ventricular walls were significantly thinner and valvular development was lacking, which suggested that miR-20b overexpression affected the normal formation of the ventricular wall and the endocardial cushion at the atrioventricular canal. The results of whole embryos in situ hybridization showed that after the overexpression of miR-20b, the expressions of the cardiac progenitor cell marker genes hand2, nkx2.5, gata4and valvular precursor cells marker genes bmp4, notch lb, versican were significantly reduced, and the expression regions of the atrioventricular specific marker genes amhc, vmhc and cmlc2ranged significantly smaller, which indicated that overexpression of miR-20b affected zebrafish heart development by inhibiting the differentiation of cardiac precursor cells. These cardiac abnormalities aforementioned had a significant effect on cardiac function ultimately, which can be proved by the results of the determination of the heart rate. Our further studies found that miR-20b influenced the cardiac and valve development by directly targeted bmprlaa, which is an key gene in bmp signalling pathway, and microinjection of bmprlaa mRNA in miR-20b overexpression embryos can partially rescue the cardiac developmental abnormalities caused by miR-20b overexpression. In summary, miR-20b play an critical role in myocardial and atrioventricular valve development by affecting bmp signaling pathway in vertebrates.