| Background: Breast carcinoma is one of the most common malignant tumors in women with a good prognosis.In recent years,the incidence of breast carcinoma is consistently rising,and the age of onset is becoming lower.Molecular genetic studies also show that the carcinogenesis of breast carcinoma is a process involved multi-gene and multi-step,in which the activation,overexpression or amplification of oncogenes and the deletion or mutation of tumor suppressor genes may play a crucial role.For the development of molecular targeted agents,it is very important to study the molecular pathogenesis of breast carcinoma and search for new biomarkers that can be used for diagnosis and prognosis judgment and new therapeutic targets.Increasing evidences indicate that epigenetic modifications,such as DNA methylation,histone modification,chromatin remodeling and non-coding RNA,are associated with the pathogenesis of breast carcinoma.Histone modification is complicated as the acetylation,methylation,phosphorylation,ubiquitination,glycosylation and other modifications may occur at the N-terminus of histone proteins.These modifications may alter the interaction between DNA and histone and the structure of chromatin,thus affecting the transcriptional regulation of genes.Histone methylation is catalyzed by histone methyltransferases(HMTases),and the lysine or arginine residues at the N-terminal of H3 and H4 histones are frequently methylated.The triple-methylation is relatively stable and can affect the epigenetic memory in the long-term.EZH2(enhancer of zeste 2),a histone methyltransferase containing a SET domain,is a catalytic subunit of Polycomb Repressive Complex 2(PRC2).EZH2 mainly catalyzes the trimethylation of H3 lysine27(H3K27me3)and represses the transcription of genes.NSD2(nuclear SET domain-containing 2)belongs to the NSD protein family and mainly catalyzes the dimethylation of H3 lysine 36(H3K36me2).Studies show that EZH2 and NSD2 are highly expressed in a variety of tumors and closely involved in tumor growth,invasion,metastasis and prognosis.A recent study suggests that the expression of NSD2 and EZH2 are correlated in prostate carcinoma.This correlation is mediated by a specific histone methyltransferase regulatory axis: EZH2 directly regulates H3K27me3,which is associated with the repression of gene transcription.EZH2,as an upstream regulator of NSD2,regulates NSD2 and H3K36me2 via micro RNA net.The EZH2-NSD2 regulatory axis highlights the complexities of dynamic changes of epigenetics in diseases and normal development.Objective: The aim of this study is to investigate the relationship between EZH2 and NSD2 and the effects on histone methylation markers(H3K27me3 and H3K36me2),and to illuminate the effcets on cell proliferation,migration,and invasion of breast carcinoma,providing a foundation for elucidating the mechanism of EZH2-NSD2 regulatory axis in the tumorigenesis and development.Methods:1.We established the breast carcinoma cell line MDA-MB-231 transfected stably with EZH2 sh RNA or NSD2 sh RNA by lentivirus.2.EZH2 was overexpressed in wild-type cells,sh NSD2 cells and negative controll cells by adenovirus-mediated transfection.The effects of EZH2/NSD2 knockdown on cell proliferation,migration and invasion were detected by CCK-8assay,scratch assay and Transwell invasion assay,respectively.3.The m RNA expression level of EZH2/NSD2 was detected by Realtime PCR.Western Blot was used to detect the protein expression levels of EZH2,H3K27me3,NSD2 and H3K36me2.4.Western Blot was used to detect the protein expression levels of EZH2,H3K27me3,NSD2 and H3K36me2.5.The effects on cell proliferation,migration and invasion were detected by CCK-8 assay,scratch assay and Transwell invasion assay,respectively.Result:1.The knockdown efficiencies of EZH2/NSD2 single-site shRNA were(68.2%,59.4%)/(82.5%,85.6%),respectively.The double-site knockdown efficiencies of EZH2/NSD2 were 74.9% / 94.5%,respectively.Due to the high efficiency of double-site interference,double-site interference was uesd to establish the cell lines with EZH2/NSD2 knockdown in this study.In cells transfected with EZH2double-site sh RNA,both EZH2 and NSD2 m RNA expression were down-regulated(P=0.005,P=0.017).NSD2 and H3K36me2 protein expression were also decreased significantly as the down-regulation of EZH2 and H3K27me3 protein expression.In cells transfected with NSD2 double-site sh RNA,NSD2 m RNA expression was down-regulated(P=0.005),NSD2 and H3K36me2 protein expression were also significantly decreased.However,the expression of EZH2 and H3K27me3 had no obvious change.2.Knockdown of EZH2 significantly reduced the cell proliferation(P<0.001),migration(P=0.001)and invasive(P=0.009).Knockdown of NSD2 also weakened the ability of cell proliferation(P=0.013),migration(P<0.001)and invasive(P=0.009).3.EZH2 m RNA expression was up-regulated by 54.3-fold(P=0.032)and NSD2 m RNA expression was also up-regulated by 1.73-fold(P=0.011)in cells overexpressed with EZH2.The protein expression of EZH2,H3K27me3,NSD2 and H3K36me2 were significantly up-regulated.4.Overexpression of EZH2 significantly increased the cell proliferation(P<0.001),migration(P<0.001)and invasion(P=0.006).5.Compared with sh NC2 cells,EZH2 m RNA expression was significantly up-regulated(P=0.002)in sh NSD2 cells overexpressed with EZH2,and EZH2 and H3K27me3 protein expression were also significantly up-regulated.But NSD2 m RNA expression was not up-regulated(P=0.993),and neither NSD2 nor H3K36me2 protein expression had no obvious change.6.Overexpression of EZH2 couldn't promote cell proliferation,migration and invasion when NSD2 was knockdown.Conclusion: In breast carcinoma cell line MDA-MB-231,EZH2 directly regulates H3K27me3 that is associated with inhibiting gene transcription,and also functions as an upstream regulator of NSD2.EZH2 also regulates the expression of NSD2 and H3K36me2 that is associated with activating gene transcription.It depends on the expression of NSD2 that EZH2 promotes cell proliferation,migration and invasion of breast carcinoma. |
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