The Study On Expression And Regulation Of Pde4d In Embryo Development And Its Impacts On Cell Proliferation And Migration

Pde4d is located on mouse chromosome13. The product of Pde4d is phosphodiesterase (PDE)4D that belongs to a large superfamily of PDEs. PDE4D hydrolyzes second-messenger cyclic adenosine monophosphate (cAMP) to serve important function in cAMP/CREB and cAMP/EPAC signaling pathway. It was reported that Pde4d exhibited paternal-origin sex bias in E9.5whole embryo and Pde4d played key roles in genetic diseases and tumorgenesis. The SNP used in the previous report was located in the intron of Pde4d, the relationship between Pde4d and embryogenesis was not clear, so the imprinting pattern of Pde4d was clarified by sequencing PCR product. In order to identify the possible role of Pde4d in embryo development, expression and regulation pattern of Pde4d were investigated in this study. Mesenchymal stem cell MS-K was used as a model to study the effect of PDE4D on cell behavior to clarify the possible function of PDE4D in embryo development.To determine the imprinting characteristics of Pde4d in embryo development, Pde4d locus was analyzed by bioinformatics. It was showed that the SNP used in the previous report was located in AK046832, which was within the the intron of Pde4d. It was suggested that AK046832could be paternally expressed. In addition, great changes of DNA methylation were happened in two CpG islands (CGI1and CGI2), suggesting putative function of the two CpG islands on imprinted expression of Pde4d locus. Imprinting analysis showed that imprinting pattern was different among Pde4d, AK046832and miR-1904, Pde4d showed paternally-biased expression in E9.5whole embryo and placneta, but biallelic expression in E15.5major tissues and placenta. AK046832and miR-1904showed the same biallelic expression during the entire process of embryogenesis. In summary, these results supported the idea that Pde4d showed dynamic pattern of imprinting expression during the embryonic development.To determine the spatiotemporal expression patterns of Pde4d in embryogenesis, Pde4d expression in embryo and placenta was studied by real-time PCR (qRT-PCR) and RNA in situ hybridization (ISH). In early embryonic stage, qRT-PCR showed gradually decreased expression level of Pde4d (2-cell to morula) and significanly decreased expression in blastula，increased expression level of Pde4d in mid-late embryonic stage (E9.5-E18.5), and placenta showed elevated expression of Pde4d at late embryonic stage. RNA ISH showed that Pde4d was mainly expressed in the nervous system and somite at middle embryonic stage (E9.5-E11.5), Pde4d was highly expressed in major organs but heart at organ formation stage (E12.5-E15.5). Pde4d expression was obviously detected in E11.5spongiotrophoblasts and labyrinth, the expression of Pde4d was decreased in labyrinth but increased in spongiotrophoblasts from E12.5-E15.5. At E16.5, decidua showed that the evaluated Pde4d expression, with the development of placenta, expression of Pde4d was detected in all three layers of placenta. The results supported the idea that Pde4d was dyanamically and ubiquitously expressed in embryo and placenta development.In order to identify the regulation factor of affecting Pde4d expression, the regulation of DNA methylaion and intronic miRNA on gene expression was studied. The result showed that CpG sites in promoter can regulate the gene expression with the tissue-specific methylation in E15.5tissues. Moreover, CpG sites in promoter with developmental stage-specific also affected the gene expression in the liver at three developmental stages. Demethylated promoter was correlated with the upregulated expression level of Pde4d in the cell treated with5-aza-cdR, which confirmed the relationship between the CpG methylation and gene expression. In addition, intronic miR-1904can target the hosted Pde4d by bioinformatics tools and luciferase reporter assay, Pde4d protein was decreased significantly in the cell overexpressed miR-1904, so intronic miR-1904could directly.regulate expression of Pde4d. By all accounts, these results showed that DNA methylation of promoter and the intronic miR-1904were important regulators of Pde4d.To get the further known about the function of Pde4d, both of short isform (PDE4D1) and long isform (PDE4D7) were studied to address the effect of PDE4D on cell proliferation and migration, respectively. The results revealed that both PDE4D1and PDE4D7significantly inhibited MS-K cell proliferation and colony formation，and both of two isforms induced the upregulation of the Cyclin D1expression to shorten the duration of G1phase and drived the cell enter S phase. In addition, the migration and spreding ability of MS-K cell were impaired due to the overexpression of two isoforms, increased Vinculin and decreased Integrin β1induced by PDE4D was one of reasons for decresed migration and spreding ability, respectively. Besides, several proliferation and migration-related genes were down regulated induced by overexpression of PDE4D. Finally, cell transplant assay in vivo showed that overexpression of PDE4D7could impaire the proliferation of MS-K cell. In summary, these results showed that PDE4D1and PDE4D7could inhibit proliferation and migration of MS-K cell in vitro and vivo.Taken together, Pde4d was a special incomplete imprinted gene which showed dynamic imprinting and extensive expression pattern during embryo development, DNA methylation of promoter and the intronic miR-1904were important regulators of Pde4d. Pde4d could affect proliferation and migration of MS-K cell. By all accounts, it is implied that Pde4d may play important roles in embryogenesis.