The Effect Of MEG8-DMR On Biological Properties Of Huh7 Cell

The DLK1–DIO3 imprinted locus on human chromosome 14q32,contains three imprinted protein-coding genes DLK1,RTL1 and DIO3 expressed from the paternally inherited chromosome and several imprinted large and small non-coding RNA genes expressed from the maternally inherited homolog,as well as numerous C/D sno RNAs and micro RNAs.Two major elements involved in regional imprinting regulation are IG-DMR and Gtl2/MEG3-DMR,which are methylated on the paternal.Studies has shownd that knonkout these DMRs on differently parental alleles effectd genes expression and embryonic development in the interval.MEG8-DMR is located in intron 2 of MEG8,the novel DMR is methylated on the maternal allele.Previous studies found that insertional mutagenesis by AAV vector or SB transposon around Meg8-DMR of mouse chromosome in this domain leaded to deregulated of adjacent genes and facilitated hepatocarcinogenesis.We assumed that MEG8-DMR might be unknown regulatory sequences on the expression of genes and the occurrence of hepatocellular carcinoma.In this study,we knockouted MEG8-DMR by CRISPR/Cas9 on the genome level of HCC cell line Huh7,focus on the effect on expression level of genes and biological properties.We constructed the Cas9-MEG8-DMR recombinant vector and transfected Huh7 cell,the results showed that MEG8-DMR could be knockedout effectively.Then we analyzed the expression of DLK1,DIO3 and IGF2 were decreased,whereas in increased expression of MEG9,the expression of mi R-370 accompanied by deregulated,no significantly change of MEG3 and MEG8.We analyzed the effect on biological properties of Huh7 cell.The result showed that knockout MEG8-DMR inhibited the proliferation and cloney formation capacity significantly,induced cells apoptosis.But there was no significantly change in cell morphology,migration ability,invasion pathways and cell cycles.Taken together,MEG8-DMR regulated the biological properties of Huh7 cell though disturb the exptessions of related genes.The results will contribute to the further investigation of regulatory mechanism and functions of MEG8-DMR.