Mechanism Of MiR-375 Overexpression-induced Cardiac Dysplasia Based On Zebrafish Model

Congenital heart disease(CHD)is the most common disease of infant birth defects in the world,which has a high incidence and brings heavy medical and economic burden to the family and society.Although a lot of research have been invested in CHD,its mechanism has not been clear.It is the result of the interaction of complex genetic factors and environmental factors.With the progress of new gene sequencing technology,especially reverse genetic technology,some new genes have been found to be related to CHD,so a series of studies have been carried out.Micro RNAs(mi RNA),as small non-coding RNAs,regulate the expression of target genes by binding to target genes,and play an important role in the growth and development of organisms and diseases,especially for the normal development of the heart.In the previous study on fetal tissue samples of ventricular septal defect(VSD)induced by spontaneous abortion,our team screened a series of differentially and highly expressed gene sequences by microarray technique,and we selected mi R-375 to conduct in-depth study.compared with the normal control group,previous studies confirmed that mi R-375 plays a key role in cardiomyocyte differentiation at the cytological level.In this study,reverse genetics technique was used to explore the important role and mechanism of mi R-375 in zebrafish cardiac development through zebrafish model.The animal model of mi R-375 overexpression was established by microinjection of different concentrations of mi R-375 mimic into zebrafish embryos,and the cardiac development of zebrafish was observed in vivo.We found that the overexpression of mi R-375 caused a series of damage to the heart development of zebrafish embryos,including slow heart rate,pericardial edema and circulatory abnormalities.At the same time,in addition to the phenotype of the heart,other organs of zebrafish are also affected to varying degrees,including brain,eyes,digestive system,bone and caudal fin,suggesting that the effects of mi R-375 overexpression on embryos involve many other systems besides the heart.Through real-time quantitative PCR,we confirmed that overexpression of mi R-375 can significantly inhibit the expression of cardiac development-related genes.Furthermore,luciferase reporter gene study confirmed that notch2 was the target gene of mi R-375,and the expression of downstream genes related to NOTCH signal pathway was also significantly suppressed.In situ hybridization showed that the expression of notch2,a key gene in heart development,was significantly down-regulated and the expression range became smaller.It was confirmed that the effect of overexpression of mi R-375 on zebrafish heart development was achieved by directly acting on the target gene notch2 and then affecting the NOTCH signal pathway.In conclusion,zebrafish can be used as a high-quality animal model in the study of cardiac development.Mi R-375 can affect the cardiac development of zebrafish through affecting the NOTCH signaling pathway by targeting notch2.