Regulation And Mechanism Of Gga-miR-302d And Gga-miR-1777 In Maintaining Phiripotency Of Chicken Embryonic Stem Cells

MicroRNA(miRNA)was an endogenous non-coding single-stranded RNA molecule of length 19~23nt(Nucleotide,nt),which was usually regulated by degrading the target mRNA or blocking the translation of the target mRNA.Studies in mice or human had shown that there were specific highly expressed miRNAs with key role in maintaining the pluripotency in embryonic stem cells,while there was relatively few in chicken embryonic stem cells.The specific highly expressed miRNA were screened from cESCs and CEFs by miRNA chip in present study,and then veritied by RT-PCR.Subsequently,the target genes of candidate miRNA were predicted and the expression level were detected by RT-PCR in blastocysts and chicken embryo fibroblasts.Finaly,the target gene of miRNA was further verifiedby dual luciferase detection system.The results showed as follows:1.Highly expressing miRAN were screened in cESCs by miRNA chip.And the expression levels of gga-miR-1777,302 d,and 1599 were 24.43,210.81 and 25.37 times respectively than those in CEFs.2.The relative expression levels of gga-miR-1777 in cBDCs were 232.90% of which in CEFs,and the relative expression levels of gga-miR-302 d and gga-miR-1777 in cBDCs were respectively 439.73% and 183.47 % of which in CEFs(P <0.05),which was consistent with the miRNA chip results.3.The relative expression levels of gga-miR-1777 in cBDCs(0h)were down-regulation of 7% and 21% of which in cBDCs at stage HH2 and at stage HH3.The relative expression levels of gga-miR-302 d in cBDCs(0h)were down-regulation of 24% and 447% of which in cBDCs at stage HH2 and at stage HH3(P<0.05).4.The potential target genes of gga-miR-1777 and gga-miR-302 d were predicted by TargetScan and miRDB database,then Atxn1 and Wdr1 were selected as candidate genes for gga-miR-1777 and gga-miR-302 d respectively.5.The relative expression levels of gene Wdr1 in cBDCs were 64.05%(P<0.01)ofwhich in CEFs.The relative expression levels of gene Atxn1 in cBDCs were 57.53%(P<0.01)of which in CEFs.The results suggested that the Atxn1 and Wdr1 might be the target gene of gga-miR-1777 and gga-miR-302 d respectively.6.Double luciferase reporter vector of WT Wdr1-3'UTR(WT Atxn1-3'UTR)or MUT Wdr1-3'UTR(MUT Atxn1-3'UTR)was transfected in 293 T cells,the result showed that the reported fluorescence value for gga-miR-302 d mimics on the Wdr1 wild type vector was up-regulation of 14%,while the reported fluorescence value for gga-miR-302 d mimics on the Wdr1 mutant vector was down-regulation of 31%.indicating that there was significant difference between them(P<0.05).The reported fluorescence value for gga-miR-1777 mimics on the Atxn1 wild type vector was up-regulation of 11%,while the reported fluorescence value for gga-miR-1777 mimics on the Wdr1 mutant vector was down-regulation of 19%,indicating that there was no significant difference between them.It showed that gga-miR-302 d was likely to regulate gene expression through the site on the3'UTR of the Wdr1 gene,whereas gga-miR-1777 was likely to have no significant interaction with the 3'UTR of the Atxn1 gene.The above results indicated that gga-miR-302 d,which is highly expressed in chicken embryonic stem cells,might maintain the pluripotency of chicken embryonic stem cells by inhibiting the expression of Wdr1.