The Research Of The Regulatory Relationship Among Pde4d Its Intronic MiR-1904 And CGI

Pde4d is the first inprinted gene located on mouse chromosome 13,it showed paternally-biased expression in E9.5 embryo,and biallelic expression in E15.5 days.Many studies have shown that Pde4 d played a key role in diseases and tumor.The product of Pde4 d is phosphodiesterase(PDE)4D that belong to superfamily of PDEs,which hydrolyzes second-messenger cyclic adenosine monophosphate(cAMP)in cells.Analyzed the Pde4 d locus by bioinformatics found that Pde4 d have 17 exons.There was a CpG islands(CGI96)in its seventh intron,and a miRNA(miR-1904)in its eighth intron,DNA methylation and non-coding RNA were very important epigenetic modifications,and played a key role in the development and disease are very important.miR-1904 is only found in mouse,and it was an intronic microRNA.The form of this "intronic miRNA-host gene" often has important biological fundation,so this study research the regulatory relationship between Pde4 d and mi R-1904.First,we use bioinformatics tools and luciferase reporter assay to ensure intronic miR-1904 can target its hosted gene in Hepa1-6 cells.We change the expression level of mi R-1904 by transfecting miR-1904 mimics and miR-1904 inhibitor,then analyzed the Pde4 d expression level by qRT-PCR and Western Blot.The result show Pde4 d was decreased in the cell when over expressed(low expressed)miR-1904 and vice versa.These result show that miR-1904 can regulate the expression of its host gene Pde4 d.CGI96 is located in the eighth intron of Pde4 d,which is very close to the upstream of miR-1904.The DNA methylation level of CGI96 in the liver and Hepa1-6 cells was analyzed.we knockout the CGI96 using CRISPR/Cas9 on the genome level of cell.Then we use analyzed tha expression levels of miR-1904 and Pde4 d.After knockout of CGI96 on Hepa1-6,the expression levels of miR-1904 and Pde4 d were significantly decreased.The result indicated that Pde4 d and miR-1904 were regulated by CGI96.In summary,the CGI96 and intronic miR-1904 in Pde4 d are important regulators of Pde4 d.The aim of this study is to investigate the regulated relationship of them and provide more data to the research of epigenetic regulation.