MiRNA-30c Target Inhibiting Cxcr4a And Regulating Embryo Endoderm Organ Formation

Objective:We discuss important issues on miR-30 c regulation of cell differentiation and embryonic patterning.Methods:1.Bioinformatics:1)The nucleotide sequences of miR-30 family members in each species were obtained from the mi RBase database(http://www.mirbase.org/), and multiple sequence alignment was performed by using the software W Clustal.2)In miR- 30 members of the family of their original transcript(pri- miR- 30) nucleotide sequence based on the system of trees by the software in MEGA5 adjacency method(neighbor joining method) calculation.2.TaqMan PCR and whole-mount in situ hybridization:Zebrafish genome DNA extraction, PCR and probe plasmid construction, digoxin tag antisense RNA probe preparation, the whole embryo in situ hybridization, miRNA expression analysis based on TaqMan PCR technology3. Microinjecting mi R-30c-duplex,microinjecting miR-30c-MO :The mRNA(egfp-cxcr4a-3’UTR mRNA、egfp-cxcr4a-3’UTR(miR-30cmut) mRNA), duplex(miR-30c-duplex)and morpholino(dre- miR-30c-MO)were injected into the embryonic cells and set the control group.4.Online miRNA target prediction analysis and whole-mount miRNA sensor assay。Results:In this study, 1.We investigated the spacial and temporal expression pattern of mi R-30 c during early developmental stages, and found that miR-30 c is maternally lowly expressed, and then ubiquitously expressed with abundant expression in mesendoerm during later gastrulation and mid-somite stage, and eventually restricted to the pronephric duct at 24 hpf. 2.Overexpression of miR-30 c reduced the midline migratory ability of mesendodermal cells, eventually displaying cardiac and viscera bifida in zebrafish. 3.Knockdown of mi R-30 cgives rise toenhanced midline migration of mesendodermal cells.4.We demonstrated that cell migration-related factor cxcr4 a is one of the potential targetof miR-30 c in this context.Conclusion:1. Preliminary study on the temporal and spatial expression patterns of miR-30 c in the early stage of embryogenesis. 2. MiR-30 c is an important regulatory factor in the formation of endodermal cells and organs to midline migration. 3. By up regulating the signal intensity of Cxcr4 a protein, the normal development of embryos can be ensured.