Successful cloning through somatic cell nuclear transfer (SCNT) faces significant challenges due to epigenetic obstacles. Recent studies have highlighted the roles of H3K4me3 and H3K27me3 as potential contributors to these obstacles. However, the underlying mechanisms remain largely unclear. In this study, we generated genome-wide maps of H3K4me3 and H3K27me3 in mouse pre-implantation NT embryos. Our analysis revealed that aberrantly over-represented broad H3K4me3 domain and H3K27me3 signal lead to increased bivalent marks at gene promoters in NT embryos compared with naturally fertilized (NF) embryos at the 2-cell stage, which may link to relatively low levels of H3K36me3 in NT 2-cell embryos. Notably, the overexpression of Setd2, a H3K36me3 methyltransferase, successfully restored multiple epigenetic marks, including H3K36me3, H3K4me3, and H3K27me3. In addition, it reinstated the expression levels of ZGA-related genes by reestablishing H3K36me3 at gene body regions, which excluded H3K27me3 from bivalent promoters, ultimately improving cloning efficiency. These findings highlight the excessive bivalent state at gene promoters as a potent barrier and emphasize the removal of these barriers as a promising approach for achieving higher cloning efficiency.
Animals ; Mice ; Histone-Lysine N-Methyltransferase/biosynthesis* ; Histones/genetics* ; Nuclear Transfer Techniques ; Female ; Gene Expression Regulation, Developmental ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Embryo, Mammalian/metabolism*

OBJECTIVETo determine the potential of SMYD3 as a therapeutic target for hepatocellular carcinoma (HCC) by potent and highly sequence-specific RNA interference (RNAi) technique.

METHODSThe mRNA of SMYD3 was detected by RT-PCR in different HCC cell lines, such as HepG2, Hep3B and SMMC7721. Recombinant SMYD3 shRNA plasmid Pgenesil-1-s was constructed and transfected into HepG2 cells, and Western blot was used to identify the down regulation of SMYD3 protein expression after transfection. MTT and flow cytometry analysis (FCM) were respectively applied to analysis cell proliferation and apoptosis. In vivo study was carried out by injecting recombinant SMYD3 shRNA plasmids into transplanted tumors of nude mice.

RESULTSThe expression of SMYD3 mRNA was abundant in HCC cell lines HepG2, Hep3B, SMMC7721, whereas none in normal hepatic cell line L-02. RNA interference was able to suppress SMYD3 expression greatly and then inhibited cell growth effectively and induced apoptosis of HepG2 cells efficiently. After injection of recombinant SMYD3 shRNA plasmid, transplanted tumors grew slowly and reduced in size and weight when compared with those of control groups (P < 0.01).

CONCLUSIONSSMYD3 plays a major role in occurrence and progress of HCC. Inhibition of SMYD3 by RNAi can induce apoptosis in HepG2 cells and suppress tumor growth in nude mice. Therefore SMYD3 could be an ideal therapeutic target for HCC.

Animals ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; pathology ; therapy ; Cell Line, Tumor ; Genetic Therapy ; methods ; Histone-Lysine N-Methyltransferase ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; genetics ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVESTo identify the inhibition effect of shRNA on the SMYD3 (SET- and MYND-domain containing protein-3) expression in hepatoma cell line HepG2 through gene silencing.

METHODSTwo reverse repeated motifs targeting on the SMYD3 mRNA sequences 267-288, 302-323 respectively, were synthesized and inserted into the mock plasmid pGenesil-1 which expressed EGFP to create recombinant plasmids pGenesil-1-s1 and pGenesil-1-s2. pGenesil-1-hk specific to no SMYD3 mRNA sequence served as a control. After transfection into HepG2 cells, RT-PCR and western blot were applied to identify the down regulation of SMYD3 expression by shRNAs.

RESULTSAll plasmids were constructed successfully. pGenesil-1-s1, pGenesil-1-s2 inhibited the mRNA and protein expression of SMYD3 in HepG2 cells. There was a significant distinction when compared with pGenesil-1-hk and pGenesil-1 (P<0.01).

CONCLUSIONShort hairpin RNAs can efficiently and specifically suppress the expression of SMYD3 in HepG2 cells.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Histone-Lysine N-Methyltransferase ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection