MicroRNA-206 Plays Roles on Somite Boundary Development through Silencing rtn4a and Its Downstream Genes

MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level by targeting the 3’-untranslated region (3’UTR) of mRNAs through a seed sequence. miR-206 is a muscle-specific microRNA and is highly expressed in skeletal muscle. It is able to regulate the differentiation and proliferation in myoblast. That reticulon4a (rtn4a) is one of miR-206 target genes is proved by Labeled miRNA pull-down assay (LAMP) which preformed in 16-hpf zebrafish embryos. Further studying shows that miR-206 knockdown or rtn4a overexpression leads to somite boundary defect where F-actin across somites within 48-hpf zebrafish embryos. It implys that miR-206 plays roles on somite boundary development through silencing rtn4a. To address this issue, we anlazyed the gene expression profile of somite cells of miR-206-knockdown or rtn4a-overexpression embryo by microarray. Compared that with gene expression profile of wild type, we found the candidate genes which may be downstream of miR-206-rtn4a pathway. After microarray analysis, we selected 4 candidate genes which were all up-regulated and 4 candidate genes which were all down-regulated in miR-206-knockdown or rtn4a-overexpression embryo for further study. Using whole-mount in situ hybridization, we further selected two candidate genes ,cxcr4a which is down-regulated and thbs3a which is down-regulated, by its expression patterns and level. Then, we observerd that somite bounday defect within cxcr4a-knockdown or thbs3a-overexrpresion zebrafish embryos. Furthermore, it was proved that cxcr4a was able to repress thbs3a mRNA expression. As mentioned above, rtn4a was silenced by miR-206 inhibated cxcr4a, which repressed thbs3a mRNA expression. In addition, we demonstrated that somite boundary defect as mentioned might result from somite boundary cells wasn’t able to differentiate to epithelium by detecting the arrangement of γ-tubulin or expression of pFAK around the defect area. Evidence thus far accumulated shows that rtn4a was silenced by miR-206 inhibiate cxcr4a and leads to repress thbs3a to regulated the epithelialization of somite boundary cells during the somite development of zebrafish embryo.