Experimental Study Of The Regulation Of EZH2 On Children Hepatoblastoma Cells

Objective:Hepatoblastoma(HB)is the most common type of malignant liver tumor in children.While outcomes have been greatly improved in the past decades,the treatment of advanced HB has remained challenging.To study the potiential diagnostic markers and therapeutic targets has become an important approach for treatment of HB.Histone modications,such as methylation and acetylation,which are important parts of epigenetic regulation,plays vital roles in the tumorgenesis of diverse tumors.Enhancer of zeste homologue 2(EZH2),responsible for the trimethylation of histone H3K27(H3K27me3),is amplified and overexpressed in a variety of cancers.However,the role of EZH2 in HB has remained to be fully elucidated.The purpose of the present study was to investigate the expression patterns of EZH2 in HB cells and to detect its potential regulatory mechanism.Methods: Initially,we performed western blotting to investigate EZH2、P27 expression in HB specimens compared with peri-tumor tissues.si RNA and lentiviral small hairpin RNA was used to inhibit EZH2 expression in HB Hep G2 and Huh6 cell lines.Western blotting and polymerase chain reaction was used to detect the effects of inhibition.We used cell counting kit-8 to determine effects of EZH2 on proliferation and flow cytometric cell cycle analysis to effects of EZH2 on cell cycle after EZH2 suppression.DZNep,a well-known histone methyltransferase inhibitor,was used to decrease EZH2 expression,and its effects on proliferation,apoptosis-inducing and colony formation were examined in HB cell lines.Human WNT signaling pathway PCR array was used to detect effects of EZH2 on gene expression of WNT signaling pathway via suppression of EZH2 in Huh6 cell line.Effects of DZNep on the WNT activity of Hep G2 and Huh6 cells were detected by Dual-luciferase Reporter Assay System.Result: EZH2 expression was significantly higher in HB specimens compared with that in peri-tumor tissues,while P27 was reduced in HB.Suppression of EHZ2 using si RNA and lentiviral small hairpin RNA inhibited HB cell proliferation,induced cell cycle arrest in G1 phase and enhanced the expression of G1/S-phase checkpoint protein P27.DZNep could inhibit the proliferation,induce apoptosis,decrease colony formation abilities of HB cells.Inhibition of EZH2 in Huh6 cell had shown to affect some genes expression of WNT pathway and in which 14 distinct changes might be candidates for further study.WNT activities were increased after suppression of EZH2 by DZNep.Conclusion: These results suggested that EZH2 may represent a potential diagnostic marker and therapeutic target for the treatment of HB.It exerts its oncogenic function at least in part by inhibiting p27 expression and thereby reducing the proliferation of HB cells by blocking G1-to-S-phase transition.DZNep is a promising therapeutic agent for HB cells,with potential to inhibit proliferation,induce apoptosis and decrease colony formation.EZH2 deletion in Huh6 cell line changed some genes expression of WNT pathway,and further studies are required to more fully elucidate the mechanisms.