EZH2 And EZH1 Play Critical Roles In The Proliferation Of Mantle Cell Lymphoma Through Distinct Manners On The Regulation Of Cell Cycle

Purpose: The aim of this study was to elucidate the dysregulation of epigenetic mechanisms in the pathogenesis of Mantle Cell Lymphoma(MCL),and to investigate the functional role of epigenetic methyltransferases EZH2 and EZH1 in MCL patients and MCL cell lines.We tried to emphasize that simultaneously targeting both EZH2 and EZH1 potentially provide a novel therapeutic option for MCL patients.Methods: The protein levels of EZH2 and EZH1 of the formalin fixed paraffin embedded(FFPE)tissues were retrospectively analysed using immunohistochemical(IHC)staining and correlated to clinical outcome by SPSS software(Version 17.0)in a cohort of MCL patients.Quantitative real-time PCR and Western Blot were applied to confirm the level of EZH2 and EZH1 in MCL cell lines which were compared to those of the Na?ve B cells.To evaluate the effect of UNC1999 on cell proliferation,apoptosis and cell cycle distribution,four MCL cell lines were treated with increasing concentration of UNC1999.In order to further gain insight into the molecular mechanisms,the gene expression profiling of Z138 and Mino cells after 8 ?M of UNC1999 treatment was performed by RNA-sequencing.Chromatin immunoprecipitation assay(Ch IP)was performed on the promoters of target genes simultaneously.To directly investigate the functional roles of EZH2 and EZH1 in the pathogenesis of MCL,Z138 cells with EZH2 and EZH1 separately knocked out using CRISPR/Cas9 system were established and further determined of the influence on the proliferation and cell cycle distrubtion.Results: Part I: Overexpression of EZH2 correlates with the overall survival of MCL patients and EZH2 and EZH1 differently express in Na?ve B cells and MCL cell lines.1.Strong nuclear EZH1 expression was seen in 17.07%(7/41)MCL specimens,whereas strong nuclear EZH2 expression was seen in 48.78%(20/41)specimens.2.EZH2 overexpression was correlated with poorer overall survival(OS)(P<0.01).EZH1 positivity,however,was not correlated with OS in our cohort.3.Compared to Na?ve B cells,all four MCL cell lines expressed different level of EZH2 and EZH1.Z138 and Mino cells,which had higher levels of EZH2 protein,showed much higher level of H3K27me3 than the rest.Part II: UNC1999 inhibits tumor cell proliferation and induces cell cycle arrest by reactivating CDKN1 C and TP53INP1 in MCL cell lines.1.UNC1999 effectively inhibited tumor cell proliferation,and there was a positive correlation between inhibition of H3K27me3 and cell growth in Z138 and Mino cells(r=0.951,P=0.049 and r=0.997,P=0.003,respectively).2.Z138 and Mino cells showed significant cell cycle arrest at G0/G1 phase upon UNC1999 treatment,and there was a negative correlation between H3K27me3 inhibition and the number of cells arrested at G0/G1 phase(r=-0.970,P=0.03 and r=-0.966,P=0.034,respectively).3.UNC1999 reactivated CDKN1 C and TP53INP1 up to 3 to 30-fold on m RNA levels along with reduced levels of H3K27me3 at their promoters.Part III: EZH2 and EZH1 exhibit different effect on the proliferation and cell cycle regulation of Z138 cells via regulation of diverse target genes 1.Lenti-CRISPR-V2 plasmids that respectively targeted EZH2 and EZH1 gene were constructed and the level of EZH2 and EZH1 were effectively knocked out.2.KO-EZH2 and KO-EZH1 cells showed different effect on the proliferation and cell cycle distribution with incubation at an initial concentration of 1x10^5/ml.3.Much more cell cycle related genes were reactivated in KO-EZH1 cells compared with that in KO-EZH2 cells.EZH2 and EZH1 regulated diverse target genes.Conclusion: 1.EZH2 could be a candidate for therapies with important clinical relevance.2.UNC1999 may provide a novel therapeutic option that needs more clinical trials evaluation in MCL patients.3.EZH2 and EZH1 simultaneously play critical roles in the proliferation of MCL through regulating different cell cycle related targets.