Male-biased miR-92 from early chicken embryonic gonads directly targets ATRX and DDX3X.

MiR-17-92 cluster consists of multifunctional miRNAs related to gonadal development in mammals. Our preliminary data showed that gga-miR-92 was male-biased in chicken embryonic gonads at E5.5 and E6.5. MiR-92(a-2) and two putative targets (ATRX and DDX3X) were highly conserved and located on mammalian Chromosome X but on autosomes in chicken. Here, we studied the expression and interaction of miR-92 and the targets (ATRX and DDX3X) in chicken embryonic gonads. What's more, male-biased miR-92 shows an opposite expression tendency with ATRX and DDX3X in eight embryonic stages and different tissues at E10.5 by qRT-PCR. To verify the regulation relationship between miR-92 and two targets, we performed dual-luciferase reporter assay in DF1, overexpression and inhibition of miR-92 in chicken embryonic fibroblasts (CEFs). The results show that miR-92 directly targets ATRX and DDX3X by binding the 3' un-translated region (3'-UTR), and the over-expression and inhibition of miR-92 negatively regulates ATRX and DDX3X. After the identification of the expression of their downstream genes (AMH and WNT4) in mRNA level, we found that there is no regulatory relationship between ATRX and DDX3X. The overall results indicate that miR-92 may perform roles in early chicken gonadogenesis by regulating the expressions of ATRX and DDX3X, respectively.