| Both birds and mammals are the genetic sex determination.But unlike the mammals,the sex determination mechanism of the birds is unclear and the upstream sex determinants need to be further explored.Studies shows that miRNAs,which play function in the regulation of posttranscription,play an important role during gonadal development.We intends to find the upstream regulatory factors associated with chicken sex determination and early gonadal development from miRNA levels.Our previous study found that the expression of miR-2954,which located on the Z chromosome in birds,in gonads and other tissues in males was significantly higher than that in females,similar to that reported in other birds.These results suggested that miR-2954 may act as a cell-autonomous determinant in the early stages of chicken sex determination and gonadal development.In this study,in order to further determine and study the function of miR-2954 in early gonadal development,the relationship between miR-2954 and targets genes which involved in gonadal development and function was verified by dual luciferase reporter system,and the miR-2954 overexpression vector was constructed.The results of this study are as follows:1.The expression of miR-2954 in CEF cells of different sex and commercial DF1 cells was detected by RT-qPCR.The results showed that The expression level of miR-2954 in male CEF cells was the highest,and was extremely significantly higher than that of female and mixed(mixed with the same amount of males and females)CEF and commercial DF1 cells,which was consistent with the higher expression of miR-2954 in males in our previous study.And the results also showed that the commercialized DF1 cells are suitable for the study of miR-2954 overexpression in the subsequent experiment.2.Three expression vectors of PLIN-2954,PSIN-eGFP-2954 and PSIN-eGFP were constructed and their effects were determined by sequence analysis and fluorescence observation.The results of quantitative PCR showed that the expression of miR-2954 in the cells transfected with PSIN-2954 and PSIN-eGFP-2954 was significantly increased,indicating that the overexpression vector of miR-2954 was constructed successfully.3.The effect of miR-2954 on DF1 cell cycle was detected by PI staining after transfection with PSIN-2954 vector.The results showed that the proportion of cells at G1 phase in transfected group was extremely significantly decreased,while the cells at S and G2 phase were significantly increased,which indicated that the overexpression of miR-2954 could promote cell proliferation.4.According to the matching of the whole mRNAs of the chicken(including 5'UTR,CDS and 3'UTR)with miR-2954 sequence and these functional analysis,four genes related to chicken gonadal development and function,AMH,DMRT1,SF1 and VLDL,were screened as candidate target genes of miR-2954,and corresponding luciferase reporter vectors of target sequences were successfully constructed.5.The results of dual luciferase reporter system showed that miR-2954 could significantly or extremely significantly inhibited the activity of SF1 and VLDL-CDS double luciferase,while the luciferase activity of AMH and DMRT1-CDS vectors did not change significantly.The results showed that SF1 and VLDL were the target genes with negative regulation with miR-2954.In conclusion,the regulatory relationship between miR-2954 and SF1 and other genes which involved in early gonadal development was preliminarily established,and the overexpression vector of miR-2954 was successfully constructed.These results has laid an important foundation for the functional analysis of miR-2954 on early gonadal differentiation and development in chicken embryos in further study. |
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