| BackgroundLymphoma is one of the most common tumors in the world.Although the incidence rate of lymphoma in China is lower than that in Western countries,it also increased significantly in recent years.However,Chinese patients with lymphoma have the poor prognosis,and the 5-year survival rate of patients is 38.3%.The 5-year relative survival rate of patients increased by less than 5% over ten years since 2015.Diffuse large B-cell lymphoma(DLBCL)is the most common type of non-Hodgkin's lymphomas,accounting for 30%-40% of non-Hodgkin's lymphoma in global countries.Most patients with DLBCL can be cured with a first-line chemoimmunotherapy regimen combining anti-CD20 antibody and anthracycline antibiotic(R-CHOP).However,30%-40% of patients are insensitive to the first-line therapy or relapse,indicating that DLBCL is a kind of tumor with Strong heterogeneity.With the development of next-generation sequencing technology,people have a new understanding of the genomic features of DLBCL.Various mutations of driver genes have been reported,and some of them have been shown to be closely associated with the prognosis,such as EZH2,MYD88,NF-?B,et al.WHO classification of lymphatic tumors emphasizes the role of the molecular biomarkers in the diagnosis,treatment guidance and prognosis judgment,so that the disease can be diagnosed more accurately.Both the changes in DNA sequences and the epigenetic modifications regulate the biodiversities.Epigenetic changes refer to changes in phenotype or gene expression without altering DNA sequences,including DNA methylation,histone methylation,histone acetylation and other covalent modifications,as well as chromatin remodeling,non-coding RNA and other non-covalent modifications.The modification of the histone tail mainly inhibit or activate gene expression by acetylating or methylating different amino acids.Chromatin changes which replicate with cell division,are critical for regulating gene transcription and expression,so they play important roles in maintaining the biological functions of the cells.In recent years,whole genome sequencing demonstrated the epigenetic disorders in almost all the malignancies.Therefore,a major focus in the molecular targeted therapy for tumors is to fully understand the epigenetic abnormal changes and regulation mechanisms and to find strategies for cancer treatment by interfering epigenetic modifications.EZH2 is one kind of histone methyltransferases(HMTases),which is an important component of polycombine inhibitor complex 2(PRC2).It can regulate chromatin remodeling and gene expression by catalyzing H3K27me3 and affecting the affinity of histone to DNA.NSD2 is a histone methyltransferase which containing a nuclear receptor SET domain.It plays a role in activating transcription by catalyzing histone methylation specially.Some scholars have found that high expression of EZH2up-regulate NSD2 indirectly via microRNA networks and promote tumorigenesis in prostate cancer.Our previous study found that both EZH2 and NSD2 are highly expressed in B-cell lymphoma,and their expression are correlated to each other.It is further verified that miRNA profiling changes aberrantly,suggesting that microRNA networks may be responsible for the expression correlation between EZH2 and NSD2 in a variety of tumors.Several inhibitors of the histone methyltransferase EZH2 have been introduced into clinical trials,but there are still a series of problems with slow advancement.The selective inhibitor of EZH2 gene,GSK2816126(GSK126),is a highly selective S-adenosylmethionine competitive inhibitor containing a 2-pyridone radical group.It can occupy the site of the co-substrate SAM to inhibit enzyme activity.It has been confirmed that GSK126 can significantly inhibit the proliferation of DLBCL cell line with gain-of-function mutation of EZH2 and effectively inhibit the growth of xenograft tumors in a mouse model.The multifactorial effect of GSK126 on the biological behavior of DLBCL cells and epigenetic mechanisms have been not yet fully clarified.In summary,small-molecule inhibitor GSK126 of EZH2 may become a potential drug for the treatment of DLBCL,and has potential applications in clinical treatment of tumors.This research puts forward an "EZH2-miRNA-NSD2 axis" regulatory pathway,and studies the effect of GSK126 on the biological behavior of DLBCL cell line.Furthermore,the mechanism of EZH2-miRNA-NSD2 regulatory axis in the anti-tumor effect of GSK126 will also be investigated,providing a new idea for molecular targeted therapy to overcoming DLBCL.Objectives1.To investigate the effects of GSK126 on proliferation,colony formation,cell cycle distribution and apoptosis of DLBCL cells.2.To investigate the mechanisms of GSK126 in regulating the biological behavior of DLBCL cells.3.To investigate the mechanism by which EZH2 regulates NSD2 via suppressing miRNA expression and to observe the effect of DNA damage repair ability in DLBCL cells.Methods1.The cell proliferation was detected by CCK-8 assay.2.The colony formation was detected by soft-agar colony formation assay.3.The cell apoptosis was detected by Hoechst33258 fluorescence staining of cell nucleus and flow cytometry.4.The cell cycle distribution was detected by flow cytometry.5.Western Blot was performed to detect the expression of EZH2,NSD2 and relative markers of histone methylation,the marker of DNA damage and DNA repair protein.6.The differentially expressed miRNAs in DLBCL were analyzed using miRNA microarray.7.The expression of relative miRNA and genes were detected by Realtime PCR.8.To verify the RNA and protein expression of related molecules in 293 T cells transfected with EZH2 shRNA transiently.Results1.GSK126 inhibited the proliferation and colony formation,induced the apoptosis and arrested cell cycle in G0/G1 phase in DLBCL cells.With the increase of drug concentration,the cell proliferation activity and colony formation ability decreased significantly,and the proportion of apoptotic cells increased.With the prolongation of treatment time,the effects of GSK126 were more obvious.2.GSK126 not only inhibited the protein expression of EZH2 and its catalyzed H3K27me3,but also down-regulated the expression of NSD2,its catalyzed H3K36me2 and H4K20me2,and DNA damage repair proteins regulated by NSD2.3.Combined with literature reports,miRNA microarray and bioinformatics prediction analysis,five miRNAs were identified as the possible intermediates of EZH2-NSD2 axis,including miR-26a-5p,miR-30c-2-3p,miR-196a-5p,miR-200c-3p and miR-622.It was further demonstrated that high expression of EZH2 may up-regulate NSD2 by supressing miR-26a-5p.4.EZH2 shRNA significantly up-regulated miR-26a-5p and down-regulated NSD2 and related histone methylation levels,affecting NSD2-mediated DNA damage repair process.Conclusions1.The inhibitor GSK126 of EZH2 can significantly inhibit the proliferation and colony formation,and induce the apoptosis and cell cycle arrest of DLBCL cells in a time-and dose-dependent manner.2.The expression of EZH2 and NSD2 are consistently,and both play the carcinogenic roles in DLBCL.3.The EZH2-miR-26a-6p-NSD2 axis functions in DLBCL: EZH2 may indirectly up-regulate NSD2 expression and the DNA damage repair mediated by NSD2 via supressing miR-26a-5p. |
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