Study On The Role Of LncRNA AU016765 In Early Embryo Development In Mice

Early preimplantation embryos need to be reprogrammed to eliminate parental imprints so that the embryos can establish their own gene expression patterns.In the process of embryo reprogramming,the study of histone modification and DNA methylation is more in-depth,and the regulation of non-coding RNA needs to be further in-depth.With the application of single-cell transcriptome sequencing,it has laid the foundation for the study of lnc RNA in embryonic development.In this study,based on the results of early single-cell transcriptome sequencing,AU016765,which may play an important role in the development of early preimplantation embryos,was screened to provide basic research for further revealing the regulatory mechanism of early preimplantation embryo development.In this study,BDF1 embryos obtained from C57BL/6J and DBA2 in vitro fertilization were used as the research object.By injecting small interfering RNA into BDF1 fertilized eggs,the AU016765 knockdown model was established and embryos were cultured in vitro.Research on knockdown embryos,by observing the phenotype of preimplantation early embryos,by quantitative PCR,RNA FISH,and immunofluorescence to detect various indicators during embryo development.Preliminary research on the role of AU016765 in early embryonic development lays the foundation for the regulatory mechanism of early embryonic development.The results of this study show:1.By analyzing the results of single-cell transcriptome sequencing,AU016765 was screened out,Using RT-q PCR and FISH to find that it exists in the cytoplasm,and is transiently asymmetrically distributed in the blastomere,and its expression level is continuously decreasing,which may participate in the activation of the zygotic genome.2.Changes in the expression of AU016765 at the 2,4 cell stage resulted in abnormal expression levels of genes related to zygotic genome activation.In the AU016765 knockdown group,Dppa2 and Zscan4 gene expression levels were down-regulated,and Dppa2 protein expression levels did not change.This indicates that AU016765 does not pass through the regulatory network mechanism of Dppa2/4,and may regulate Zscan4 through other pathways,thereby participating in the activation of the zygotic genome.3.Compared with the negative control,knockdown of AU016765 inhibited the transcriptional activity of 4-cell stage embryos,resulting in specific gene expression patterns in different blastomeres at 4-cell stage,suggesting that it may have an impact on the fate determination and differentiation potential of different blastomeres.4.In the 2,4 cell stage,the expression of AU016765 changes,which has no effect on the expression of genes related to blastocyst development,but the Oct4 protein expression level is high in the early stage of the 4 cell;The protein expression level of Sox17 will be lower in 2 cells and 4 cells later.After knocking down AU016765,the embryo can still develop to the blastocyst stage,and the expression of Nanog,Sox17,Cdx2,and Oct4 is not affected,but the expression level of Oct4 protein is significantly increased.This indicates that AU016765 mainly regulates the expression of Oct4 during embryonic development.5.Knock down AU016765 to reduce embryo implantation rate and cause a certain probability of fetal malformation.In summary,AU016765 is expressed in specific time and space in preimplantation early embryos and plays an important role in the development of preimplantation embryos.AU016765 does not use the Dppa2/4 regulatory network pathway.It may regulate the expression of Zscan4 through other means and participate in the activation of the zygotic genome.AU01676 regulates the expression of Oct4 and Dppa2 proteins.The expression changes of AU016765 in the early stage affect the changes in the molecular level of preimplantation embryos,thereby affecting the development of postimplantation embryos.