Functional Studies Of MiR-200 Cluster On Chromosome 23 In The Growth Regulation Of Female Zebrafish And Yellow Catfish

In the fields of genetics and breeding and developmental biology,revealing regulatory mechanisms of fish during development has always been a key research direction,genome-wide micro RNA regulation is one of the hotspots of the direction.In this study,model fish zebrafish(Danio rerio)and aquatic economic fish yellow catfish(Pelteobagrus fulvidraco)were selected as research objects to preliminarily explore the molecular mechanisms of miR-200 family in regulating growth of fish.Studies have shown that after knocking out the miR-200 cluster on chromosome 23(chr23-miR-200)in zebrafish,the body weight of homozygous female fish is significantly higher than that of wild-type female fish,while the body weight of homozygous male fish and wild-type zebrafish has no significant difference.The differences indicate that the miR-200 family may plays an important role in the regulation of fish growth.However,whether the miR-200 family regulates development and growth by targeting certain growth-related genes is unknown.Our laboratory has previously found that stat5 b is an important gene in fish development and growth.Here,we explored the expression of miR-200 family members and stat5 b in livers of wild-type female and male zebrafish.We found that in the liver the expression levels of chr23-miR-200 family members including miR-200 a,miR-200 b and miR-429 a were significantly higher in male zebrafish than that of females,while the expression level of stat5 b in females was higher than that in males.These results indicated that miR-200 potentially regulates the expression of stat5 b.To reveal the systematic regulation of miR-200,we further took chr23-miR-200 knockout and wild-type zebrafish as research models to compare the expression of development and growth-related genes stat5 b,beclin1 and gh in the livers of female and male fish.Results showed that in both male and female fish,the expression levels of the three growth-related genes were significantly higher in chr23-miR-200 knockout than in the wild type.Through online prediction,sites in the core sequence of 3’UTR of stat5 b gene can be potentially targeted by miR-200 a,miR-200 b and miR-429 a.We confirmed the targeting of chr23-miR-200 members to stat5 b m RNA using dual-luciferase reporter assay system.In addition,we further explored the function of chr23-miR-200 members in yellow catfish.Sexual dimorphism of yellow catfish indicates that the growth rate of males is higher than that of females,so it is a good material for studying growth regulation of miR-200.We found that in the liver of wild-type yellow catfish,the expression level of miR-200 family including miR-200 a and miR-200 b was significantly higher in females than that in males,while stat5 b level in males was significantly higher than that of females.Also,using the online prediction,we found the potential binding sites of miR-200 a and miR-200 b in the core sequences of 3’UTR of the stat5 b gene.Through the dual-luciferase reporter assay system in vitro,we confirmed that miR-200 a and miR-200 b can target stat5 b m RNA by binding to its3’UTR.In order to further reveal the role of chr23-miR-200 family in the growth regulation of yellow catfish,we used CRISPR/Cas9 gene-editing technology to knock out the two precursors of miR-200 a and miR-200 b on chromosome 23 in vivo.The results of PCR amplification and sequencing showed that the knockout was successful,and the F0 generation of miR-200 knockout yellow catfish has been obtained and cultured.The F0 knockout of yellow catfish would provide a very important experimental material for further study on revealing the regulatory molecular mechanisms of the miR-200 family during the development and growth.In summary,miR-200 family on chromosome 23 plays a very important role in the regulation of development and growth in zebrafish and yellow catfish,and its regulation is implemented by targeting stat5 b gene.This study can provide a theoretical basis for further analysis of the growth regulation mechanism of the model species zebrafish and the economic fish yellow catfish.