The Expression Of EZH2 In Neuroblastomas And Its Relative Studies

Background:Neuroblastoma(NB) is the most common extracranial solid tumor in children.Patients with early-stage, low-risk disease have achieved favorable therapeutic effect. But for the patients with advanced stage disease, the five-year survival remains around 50% despite the combination of surgery, high-dose chemotherapy, radiotherapy, autologous stem cell transplantation and other treatment. It is difficult to further improve the effects of treatment by simply expanding operation or increasing the intensity of radiotherapy and chemotherapy. Molecular targeted therapy with higher specificity and sensitivity is to be the main treatment in the future. Therefore it is important to search for new and effective molecular targets for NB. EZH2(enhancer of zeste homolog 2, EZH2) is the core catalytic element of polycomb repressor complex 2(PRC2). It regulates the transcription of target genes by promoting the methylation of histone H3. Several studies have shown that overexpression of EZH2 indicated poor prognosis in a variety of cancers. And EZH2 is found to silence the tumor suppressor genes such as CASZ1, CLU, RUNX3 and NGFR in NB cells at epigenetic level, suggesting that EZH2 can be used as a potential molecular target of NB.Objective:The purpose of this study is to detect the expression of EZH2 protein in NB tissues and analyze the relationship between the expression of EZH2 and the clinicopathological factors, as also and the survival prognosis of NB patients. In addtion,we wanted o explore the effects of EZH2 on the carcinogenesis and development of NB by using the NB cell lines.Methods and results:1. The expression of EZH2 protein in neuroblastoma tissueMethods: Detect the expression of EZH2 in NB tissues of 74 cases by immunohistochemistry.Results: 2 cases with negative expression of EZH2, 21 cases with weakly positive EZH2, 31 cases with positive expression of EZH2, 20 cases with strongly positive expression of EZH2, namely 23 cases with weak expression of EZH2 and 51 cases with strong expression.2. The relationship between the expression of EZH2 and the clinicopathological features and prognosis of NB patients.Methods:Analyze the relationship between the expression of EZH2 and the clinicopathological features,as alsoand prognosis of NB patients by SPSS19.0 statistical software.Results: NB patients with strong expression of EZH2 is positively correlated with advanced stages(X2=6.931,P=0.008), distant metastasis(X2=5.722,P=0.017)and strong expression of Ki67(X2=29.827,P=0.000), and may negatively correlated with VGPR or better treatment effects, P value is close to 0.05(X2=4.045,P=0.052). The analysis of EZH2 expression and relapse in patients indicated that patients with strong expression of EZH2 is positively correlated with relapse, but the P value is greater than 0.05(X2=2.699,P=0.100). There was no significant correlation between the expression of EZH2 with age, sex,primary site and histological type(P>0.05). 3. The impact of EZH2 on the biological behavior of NB cellsMethods: Establish NB cell lines with stable overexpression or downregulated EZH2 by lentivirus. Detect the effects of EZH2 on proliferation and migration of NB cells by MTT, Ed U proliferation test and transwell assays.Results:1) NB cell lines-SK-N-BE(2)-EZH2-COPGFP and EZH2 SK-N-BE(2)-shEZH2-74、SK-N-BE(2)-sh EZH2-77 were successfully established and Sequentially verified by Western blotting.2) SK-N-BE(2) cells with EZH2 overexpression possessed greater ability of proliferation and migration compared with control and blank group;3) After downregulating the expression of EZH2 in SK-N-BE(2) cells by using small interfering RNA, the ability of proliferation and migration of the cells were significantly reduced compared with the control and blank group.4. The influence of EZH2 on the differentiation of NB cellsMethods: Investigate the effect of EZH2 on NB differentiation through inducing NB cells differentiation by retinoic acid(RA) and 5-Bromodeoxyuridine(Brdu).Results:1) SK-N-BE(2) cells showed obvious neuronal differentiation with visible andintertwined axons wire, and more smaller,gathered cells can be seen, and slower growth after inducing by RA.2) There was no obvious change in the morphology and the cell growth of SK-N-BE(2) cell after inducing by Brdu. However, the number of cells with large flat morphology(similar to stromal cells) increased gradually.3) Western bloting showed that CD133, EZH2 and Vimentin protein levels were negatively correlated with the intensity of the RA induction. On the contrary, EZH2 and Vimentin protein levels gradually increasedafter stimulation by Brdu, but the CD133 protein levels had no significant changes. The results suggested that EZH2 may play a direct role incell differentiation induced by RA and Brdu.Conclusion1) EZH2 overexpression in NB children is a cancer-promoting factor in NBs.2) Cell experiments showed that EZH2 can promote the proliferation and the migrationof SK-N-BE(2) cells.3) The expression of EZH2 protein in SK-N-BE(2) cells changed after RA and Brdu stimulation, suggesting that EZH2 may involve in the differentiation process of NB cells.