抑制EZH2表达对宫颈癌细胞凋亡的影响及机制
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摘要
目的观察抑制人类Zest同源增强子(EZH2)表达对宫颈癌细胞凋亡及凋亡相关蛋白表达的影响。方法选择人宫颈癌细胞系Siha和C33A细胞,采用MTT法确定EZH2抑制剂3-去氮腺嘌呤(DZNep)在两种细胞中的半数抑制浓度(IC_(50))。将两种细胞分为DMSO组、1/2 IC_(50)组和IC_(50)组,分别以DZNep终浓度0、3、6μmol/L处理Siha细胞各组,以0、2、4μmol/L处理C33A细胞各组。培养48 h后收集各组细胞,采用流式细胞仪检测细胞早期凋亡情况,采用Western blotting法检测EZH2蛋白、抗凋亡蛋白B淋巴细胞瘤-X基因长片段(Bcl-xL)、促凋亡蛋白Bcl-2家族细胞凋亡相互作用因子(Bim)、半胱氨酸-天冬氨酸蛋白酶3(Caspase-3)表达。结果在Siha、C33A细胞中,1/2 IC_(50)组、IC_(50)组早期凋亡率均高于DMSO组,IC_(50)组早期凋亡率均高于1/2 IC_(50)组(P均<0. 05)。IC_(50)组EZH2蛋白表达均低于1/2 IC_(50)组、DMSO组(P均<0. 05); IC_(50)组Bcl-xL蛋白表达均低于1/2 IC_(50)组、DMSO组,Bim和Caspase-3蛋白表达均高于1/2 IC_(50)组、DMSO组(P均<0. 05)。结论利用DZNep抑制宫颈癌细胞EZH2的表达后,能够诱导宫颈癌细胞早期凋亡,其机制可能与下调Bcl-xL蛋白表达并上调Bim和Caspase-3蛋白表达有关。
Objective To observe the effect of inhibiting the expression of human Zest homologous enhancer( EZH2)on the apoptosis and apoptosis-related protein expression in cervical cancer cells. Methods Human cervical cancer cell lines Siha and C33 A were selected,and we determined the half inhibitory concentration( IC_(50)) of EZH2 inhibitor DZNep( 3-denitrified adenine) in these two kinds of cells by MTT. These two kinds of cells were divided into the DMSO group,1/2 IC_(50) group and IC_(50) group. Siha cells were treated with 0,3,and 6 μmol/L DZNep,and C33 A cells were treated with the concentrations of 0,2,and 4 μmol/L,respectively. Cells in each group were collected after 48 hours of culture. The apoptosis was detected by flow cytometry. The expression of EZH2 protein,anti-apoptotic protein Bcl-xL,apoptotic protein Bim and Caspase-3 were detected by Western blotting. Results In Siha and C33 A cells,the early apoptotic rates in the1/2 IC_(50) group and IC_(50) group were higher than that in the DMSO group( both P < 0. 05),and the early apoptotic rate in the IC_(50) group was higher than that in the 1/2 IC_(50) group( P < 0. 05). The expression of EZH2 protein in the IC_(50) group was lower than that in the 1/2 IC_(50) group and DMSO group( both P < 0. 05); the expression of Bcl-xL protein in the IC_(50) group was lower than that in the 1/2 IC_(50) group and DMSO group,and the expression levels of Bim and Caspase-3 protein in the IC_(50) group and DMSO group were higher than those in the 1/2 IC_(50) group and DMSO group( both P < 0. 05). Conclusion Inhibition of EZH2 expression by DZNep can induce early apoptosis of cervical cancer cells,which may be related to down-regulation of Bcl-xL expression and up-regulation of Bim and Caspase-3 expression.
Objective To observe the effect of inhibiting the expression of human Zest homologous enhancer( EZH2)on the apoptosis and apoptosis-related protein expression in cervical cancer cells. Methods Human cervical cancer cell lines Siha and C33 A were selected,and we determined the half inhibitory concentration( IC_(50)) of EZH2 inhibitor DZNep( 3-denitrified adenine) in these two kinds of cells by MTT. These two kinds of cells were divided into the DMSO group,1/2 IC_(50) group and IC_(50) group. Siha cells were treated with 0,3,and 6 μmol/L DZNep,and C33 A cells were treated with the concentrations of 0,2,and 4 μmol/L,respectively. Cells in each group were collected after 48 hours of culture. The apoptosis was detected by flow cytometry. The expression of EZH2 protein,anti-apoptotic protein Bcl-xL,apoptotic protein Bim and Caspase-3 were detected by Western blotting. Results In Siha and C33 A cells,the early apoptotic rates in the1/2 IC_(50) group and IC_(50) group were higher than that in the DMSO group( both P < 0. 05),and the early apoptotic rate in the IC_(50) group was higher than that in the 1/2 IC_(50) group( P < 0. 05). The expression of EZH2 protein in the IC_(50) group was lower than that in the 1/2 IC_(50) group and DMSO group( both P < 0. 05); the expression of Bcl-xL protein in the IC_(50) group was lower than that in the 1/2 IC_(50) group and DMSO group,and the expression levels of Bim and Caspase-3 protein in the IC_(50) group and DMSO group were higher than those in the 1/2 IC_(50) group and DMSO group( both P < 0. 05). Conclusion Inhibition of EZH2 expression by DZNep can induce early apoptosis of cervical cancer cells,which may be related to down-regulation of Bcl-xL expression and up-regulation of Bim and Caspase-3 expression.
引文
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[14]宁向红,郭蓉,韩磊,等.DZNep通过上调miR-200c表达延缓MGC-803胃癌细胞侵袭和迁移过程[J].生理学报,2015,67(1):83-89.
[15]Liu S,Liu F,Huang W,et al.MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates ESCC tumor growth[J].Oncotarget,2017,9(3):3365-3378.
[16]Zingg D,Arenas-Ramirez N,Sahin D,et al.The histone methyltransferase Ezh2 controls mechanisms of adaptive resistance to tumor immunotherapy[J].Cell Rep,2017,20(4):854-867.
[17]Von Roretz C,Lian XJ,Macri AM,et al.Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis[J].Cell Death Differ,2013,20(1):154-168.
[18]Chang KT,Anishkin A,Patwardhan GA,et al.Ceramide channels:destabilization by Bcl-xL and role in apoptosis[J].Biochim Biophys Acta,2015,1848(10 Pt A):2374-2384.
[19]Zaanan A,Okamoto K,Kawakami H,et al.The Mutant KRASgene up-regulates BCL-XL protein via STAT3 to confer apoptosis resistance that is reversed by BIM protein induction and BCL-XLantagonism[J].J Biol Chem,2015,290(39):23838-23849.
[2]Benard VB,Thomas CC,King J,et al.Vital signs:cervical cancer incidence,mortality,and screening United States,2007-2012[J].MMWR Morb Mortal Wkly Rep,2014,63(44):1004-1009.
[3]Wu J,Sun H,Wang S,et al.Trend in relative survival in squamous cervical cancer by decade from 1983 to 2012:a period analysis[J].Cancer Manag Res,2018,10:3177-3191.
[4]Sun CC,Li SJ,Li G,et al.Polycomb group(Pc G)proteins and human cancers:multifaceted functions and therapeutic implications[J].Med Res Rev,2015,35(6):1220-1267.
[5]Vlkel P,Dupret B,Le Bourhis X,et al.Diverse involvement of EZH2 in cancer epigenetics[J].Am J Transl Res,2015,7(2):175-193.
[6]Daures M,Idrissou M,Judes G,et al.A new metabolic gene signature in prostate cancer regulated by JMJD3 and EZH2[J].Oncotarget,2018,9(34):23413-23425.
[7]Girard N,Bazille C,Lhuissier E,et al.3-Deazaneplanocin A(DZNep),an inhibitor of the histone methyltransferase EZH2,induces apoptosis and reduces cell migration in chondrosarcoma cells[J].PLo S One,2014,9(5):e98176.
[8]Pan YM,Wang CG,Zhu M,et al.STAT3 signaling drives EZH2transcriptional activation and mediates poor prognosis in gastric cancer[J].Mol Cancer,2016,15(1):79.
[9]Hu G,Gupta SK,Troska TP,et al.Long non-coding RNA profile in mantle cell lymphoma identifies a functional lncRNA ROR1-AS1associated with EZH2/PRC2 complex[J].Oncotarget,2017,8(46):80223-80234.
[10]Januario T,Ye X,Bainer R,et al.PRC2-mediated repression of SMARCA2 predicts EZH2 inhibitor activity in SWI/SNF mutant tumors[J].Proc Natl Acad Sci USA,2017,114(46):12249-12254.
[11]Xu B,Konze KD,Jin J,et al.Targeting EZH2 and PRC2 dependence as novel anticancer therapy[J].Exp Hematol,2015,43(8):698-712.
[12]Wang D,Ding L,Wang L,et al.LncRNA MALAT1 enhances oncogenic activities of EZH2 in castration-resistant prostate cancer[J].Oncotarget,2015,6(38):41045-41055.
[13]Hibino S,Saito Y,Muramatsu T,et al.Inhibitors of enhancer of zeste homolog 2(EZH2)activate tumor-suppressor microRNAs in human cancer cells[J].Oncogenesis,2014,26(3):e104.
[14]宁向红,郭蓉,韩磊,等.DZNep通过上调miR-200c表达延缓MGC-803胃癌细胞侵袭和迁移过程[J].生理学报,2015,67(1):83-89.
[15]Liu S,Liu F,Huang W,et al.MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates ESCC tumor growth[J].Oncotarget,2017,9(3):3365-3378.
[16]Zingg D,Arenas-Ramirez N,Sahin D,et al.The histone methyltransferase Ezh2 controls mechanisms of adaptive resistance to tumor immunotherapy[J].Cell Rep,2017,20(4):854-867.
[17]Von Roretz C,Lian XJ,Macri AM,et al.Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis[J].Cell Death Differ,2013,20(1):154-168.
[18]Chang KT,Anishkin A,Patwardhan GA,et al.Ceramide channels:destabilization by Bcl-xL and role in apoptosis[J].Biochim Biophys Acta,2015,1848(10 Pt A):2374-2384.
[19]Zaanan A,Okamoto K,Kawakami H,et al.The Mutant KRASgene up-regulates BCL-XL protein via STAT3 to confer apoptosis resistance that is reversed by BIM protein induction and BCL-XLantagonism[J].J Biol Chem,2015,290(39):23838-23849.