Zeste基因增强子同源物2通过调控Wnt/β-catenin信号通路影响脑胶质瘤细胞凋亡
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摘要
目的:探讨zeste基因增强子同源物2(EZH2)通过调控Wnt/β-catenin信号通路对脑胶质瘤细胞凋亡的影响。方法:以RT-q PCR和Western blot检测胶质瘤U87、H4和U251细胞及正常人脑星形细胞(NHA)中EZH2的表达水平。在H4细胞中转染EZH2 siRNA和siRNA control,MTT测定细胞活力,流式细胞术测定细胞凋亡,分光光度计法检测caspase-3的活性,Western blot检测Wnt/β-catenin信号通路中关键蛋白β-连环蛋白(β-catenin)和下游靶分子c-Myc的蛋白表达。用Wnt/β-catenin信号通路激活剂处理转染EZH2 siRNA的H4细胞,流式细胞术测定细胞凋亡,Western blot测定β-catenin和c-Myc的表达。结果:胶质瘤U87、H4和U251细胞中EZH2的mRNA和蛋白水平均明显高于NHA(P<0.05),并且H4细胞中EZH2 mRNA和蛋白水平高于U87和U251细胞(P<0.05)。EZH2 siRNA可以明显下调H4细胞中EZH2的mRNA和蛋白水平。EZH2表达下调后的H4细胞从48 h开始细胞活力降低,并且细胞凋亡率也明显升高,细胞中caspase-3活性也明显升高(P<0.05),同时EZH2表达下调还可以抑制β-catenin和c-Myc的表达。Wnt/β-catenin信号通路的激活剂可以减少EZH2诱导的H4细胞凋亡,降低细胞中caspase-3的活性。结论:EZH2在脑胶质瘤细胞中过度表达,下调其表达可以通过抑制Wnt/β-catenin信号通路的激活而诱导胶质瘤细胞凋亡。
AIM: To investigate the effect of enhancer of zeste homolog 2( EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87,H4 and U251 and normal human astrocytes( NHA) was detected by RT-q PCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway,the apoptosis rate was measured by flow cytometry,and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87,H4 and U251 were significantly higher than those in NHA( P < 0. 05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells( P < 0. 05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells,the apoptotic rate was significantly increased,and the activity of caspase-3 was significantly increased in the cells( P < 0. 05).Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2,and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.
AIM: To investigate the effect of enhancer of zeste homolog 2( EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87,H4 and U251 and normal human astrocytes( NHA) was detected by RT-q PCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway,the apoptosis rate was measured by flow cytometry,and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87,H4 and U251 were significantly higher than those in NHA( P < 0. 05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells( P < 0. 05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells,the apoptotic rate was significantly increased,and the activity of caspase-3 was significantly increased in the cells( P < 0. 05).Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2,and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.
引文
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[25]Jung HY,Jun S,Lee M,et al.PAF and EZH2 induce Wnt/β-catenin signaling hyperactivation[J].Mol Cell,2013,52(2):193-205.
[26]Chen Q,Zheng PS,Yang WT.EZH2-mediated repression of GSK-3βand TP53 promotes Wnt/β-catenin signalingdependent cell expansion in cervical carcinoma[J].Oncotarget,2016,7(24):36115-36129.
[2]Ceccarelli M,Barthel FP,Malta TM,et al.Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma[J].Cell,2016,164(3):550-563.
[3]Wang YL,Wang YT,Li JF,et al.CRNDE,a long-noncoding RNA,promotes glioma cell growth and invasion through m TOR signaling[J].Cancer Lett,2015,367(2):122-128.
[4]Tang SH,Huang HS,Wu HU,et al.Pharmacologic down-regulation of EZH2 suppresses bladder cancer in vitro and in vivo[J].Oncotarget,2014,5(21):10342-10355.
[5]Kleer CG,Cao Q,Varambally S,et al.EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells[J].Proc Natl Acad Sci U S A,2003,100(20):11606-11611.
[6]Wu Z,Qi W,Liang W,et al.Combined aberrant expression of Bmi1 and EZH2 is predictive of poor prognosis in glioma patients[J].J Neurol Sci,2013,335(1-2):191-196.
[7]Samaei NM,Yazdani Y,Alizadeh-Navaei R,et al.Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1enzyme in colorectal cancer[J].J Biomed Sci,2014,21(1):1-8.
[8]Liu YL,Gao X,Jiang Y,et al.Expression and clinicopathological significance of EED,SUZ12 and EZH2mRNA in colorectal cancer[J].J Cancer Res Clin Oncol,2015,141(4):661-669.
[9]V9lkel 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.
[10]Zhang K,Sun X,Zhou X,et al.Long non-coding RNA HOTAIR promotes glioblastoma cell cycle progression in an EZH2 dependent manner[J].Oncotarget,2015,6(1):537-546.
[11]Xu K,Wu ZJ,Groner AC,et al.EZH2 oncogenic activity in castration-resistant prostate cancer cells is polycomb-independent[J].Science,2012,338(6113):1465-1469.
[12]Mc Cabe MT,Ott HM,Ganji G,et al.EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations[J].Nature,2012,492(7427):108-112.
[13]Hoffmann MJ,Engers R,Florl AR,et al.Expression changes in EZH2,but not in BMI-1,SIRT1,DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer[J].Cancer Biol Ther,2007,6(9):1399-1408.
[14]Hyland PL,Mcdade SS,Mccloskey R,et al.Evidence for alteration of EZH2,BMI1,and KDM6A and epigenetic reprogramming in human papillomavirus type 16 E6/E7-expressing keratinocytes[J].J Virology,2011,85(21):10999-11006.
[15]Xie L,Zhang Z,Tan Z,et al.MicroRNA-124 inhibits proliferation and induces apoptosis by directly repressing EZH2 in gastric cancer[J].Mol Cell Biochem,2014,392(1-2):153-159.
[16]Srivastava SK,Bhardwaj A,Arora S,et al.MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and-independent pathways[J].Br J Cancer,2015,113(4):660-668.
[17]靳冬冬.EZH2抗卵巢癌失巢凋亡的研究[D].武汉:华中科技大学,2016.
[18]Fiskus W,Buckley K,Rao R,et al.Panobinostat treatment depletes EZH2 and DNMT1 levels and enhances decitabine mediated de-repression of Jun B and loss of survival of human acute leukemia cells[J].Cancer Biol Ther,2009,8(10):939-950.
[19]许兵,金红婷,王萧枫,等.补肾活血含药血清对成骨细胞经典Wnt/β-catenin通路的影响研究[J].中国骨伤,2015,28(6):553-558.
[20]倪明立,谢玲,秦贝贝.Wnt/β-catenin信号通路在非小细胞肺癌组织的表达及意义[J].广东医学,2016,37(15):2299-2301.
[21]Guo J,Fu Z,Wei J,et al.PRRX1 promotes epithelialmesenchymal transition through the Wnt/β-catenin pathway in gastric cancer[J].Med Oncol,2015,32(1):393-405.
[22]Chang CJ,Yang JY,Xia W,et al.EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-β-catenin signaling[J].Cancer Cell,2011,19(1):86-100
[23]Cheng AS,Lau SS,Chen Y,et al.EZH2-mediated concordant repression of Wnt antagonists promotesβ-catenindependent hepatocarcinogenesis[J].Cancer Res,2011,71(11):4028-4039.
[24]Yuan JB,Yang LY,Tang ZY,et al,Down-regulation of EZH2 by RNA interference inhibits proliferation and invasion of ACHN cells via the Wnt/β-catenin pathway[J].Asian Pac J Cancer Prev,2012,13(12):6197-6201.
[25]Jung HY,Jun S,Lee M,et al.PAF and EZH2 induce Wnt/β-catenin signaling hyperactivation[J].Mol Cell,2013,52(2):193-205.
[26]Chen Q,Zheng PS,Yang WT.EZH2-mediated repression of GSK-3βand TP53 promotes Wnt/β-catenin signalingdependent cell expansion in cervical carcinoma[J].Oncotarget,2016,7(24):36115-36129.