Study On The Mechanism Of Meg8-DMR Deletion Enhancing Migration And Invatsion Of MLTC-1

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three paternally expressed protein coding genes(Dlk1,Rtl1 and Dio3)and many noncoding RNAs expressed by the maternal parent.These genes play a key role in embryonic development,genetic diseases and human cancer.The three known paternal methylation differentially methylated regions(DMR)in this domain are Dlk1-DMR,IG-DMR and Gtl2-DMR,which play an important role in the imprinted expression regulation of genes in this domain.Meg8-DMR is the first maternally methylated DMR found in this domain.However,the target and biological function of this region are still unknown.In this study,using CRISPR/Cas9 technology,we investigated the effects of Meg8-DMR deletion on gene expression and biological function of MLTC-1.To understand the function of Meg8-DMR and its target genes,this study analyzed the functions of Meg8-DMR and its internal components,and established Meg8-DMR deletion MLTC-1 cell lines.Using bioinformatics methods,the Meg8-DMR was divided into two possible functional components,CTCF binding sites and GGCG repeats.The results of the enhancer-blocking assay showed that the mouse and human Meg8-DMR all had insulator activity dependent on CTCF binding sites.In order to further study the function of Meg8-DMR and its internal components,we constructed CRISPR/Cas9 knockout vectors,which can knock-out the Meg8-DMR,CTCF binding sites and GGCG repeats.Furthermore,the MLTC-1 cell lines with knockout of Meg8-DMR and its internal components were established by using the constructed knockout vector and finite dilution method,and were named Meg8-DMR-KO,CTCFBSKO and(GGCG)n-KO,respectively.In order to clarify the effect of Meg8-DMR deletion on gene expression,RNA-seq and RT-PCR were used to analyze and verify the differentially expressed genes when Meg8-DMR and its internal components were deleted.The results showed that Meg8-DMR-KO and CTCFBS-KO caused a large number of gene expression down-regulation on chromosome 12,especially the protein coding genes Dlk1 and Rtl1 in the Dlk1-Dio3 imprinted domain.KEGG and GO analysis showed that the co-differentially expressed genes in Meg8-DMR-KO and CTCFBS-KO were enriched on the MAPK signaling pathway and the cytoskeleton.To clarify the involvement of Meg8-DMR in the biological process of MLTC-1 cells and its mechanism,this study examined the effects of Meg8-DMR deletion on the MLTC-1 cells.We also clarified mechanism of Meg8-DMR deletion enhanced the migration and invasion of MLTC-1 cells by molecular technology.The deletion of Meg8-DMR and CTCF binding sites enhanced the migration and invasion of MLTC-1 cells,and inhibited the proliferation and colony formation of MLTC-1 cells.Further,by rescuing the expression of Dlk1 in Meg8-DMR-KO,it was proved that deletion of Meg8-DMR could enhance the migration and invasion of MLTC-1 cells by reducing the expression of Dlk1,then activating the Notch1-Rho C-MAPK/ERK signaling pathway.The methylation analysis results showed that the deletion of Meg8-DMR did not affect the methylation of Dlk1-DMR,IG-DMR and Gtl2-DMR,which indicated that in this case,the deletion of Meg8-DMR itself regulated gene expression and affected the biological function of MLTC-1.In summary,this study found that the Meg8-DMR was a conserved insulator in human and mice and depended on the CTCF binding sites.The regulatory effect of Meg8-DMR on gene expression in and out of the Dlk1-Dio3 imprinted domain was clarified.Deletion of Meg8-DMR reduced the expression of paternally expressed genes while activating the maternally expressed genes in this domain,and activated the Notch1-Rho C-MAPK/ERK signaling pathway to enhance the migration and invasion of MLTC-1 cells by inhibiting the expression of the target gene Dlk1,in which the CTCF binding sites played a key role.All the results proved that Meg8-DMR was a long-range regulatory element depending on CTCF binding sites.In conclusion,this paper expends the current understanding of DMRs in the Dlk1-Dio3 imprinted domain,and provides a basis for further research on the regulation mechanism of gene expression in the Dlk1-Dio3 domain.