The Expression Of EZH2 And MiR-127 In Chinese Ewing Sarcoma Patients And The Correlation To Prognosis

[Background and Objective]Ewing’s sarcoma is the second most common primary malignant bone tumor in childhood and adolescents,the mean age at diagnosis is 15 year and it belongs to a group of neuroectodermal tumors known as Ewing’s sarcoma family of tumors(ESFT). Clinically, Ewing’s sarcoma is a highly metastatic tumor with around 25% of patients metastasis at the time of diagnosis. In addition, nearly one third of patients will relapse after an initial clinical remission and patients who have metastatic or relapsed EWS have 5-year event free survival rates of only 10%-20%. Thus, a better understanding of the pathological mechanisms about this malignancy is critical to the development of prognositic biomarkers and novel therapies.The Enhancer of Zeste Homologue 2(EZH2) gene, a key member of the Polycomb Group(pcG) gene and play an important role in cancer. EZH2 contains a highly conserved SET domain to provide histone methyltransferase (HMTs) activity for methylating lysine 27 on histone 3(H3K27), subsequent PRC1 components are recruited to get together at specific gene loci resulting in silence of genes, including anti-oncogene and so on. Recent reasearchs have shown that EZH2 is frequently overexpression in several human tumors such as prostatic carcinoma, breast cancer, bladder cancer, hepatocellular carcinoma, Colorectal cancer, gastric cancer and so on. In a foreign country, the expression and clinical significance of EZH2 has been reported in Ewing’s sarcoma, but there is a little reported in Chinese patients.microRNAs(miRNAs) are a group of endogenous small non-coding RNAs that are approximately 18-25 nucleotides in length that play a critical role in the regulation of gene expression. By binding to the complementary sequences in the 3’untranslated regions (3’-UTR) of their targets, miRNAs play an important role in cancer. miRNAs may function not only as oncogenes but also as tumor suppressors, further implicating their roles as therapeutic targets.The purpose of this research is to detect the differential expression of microRNAs in Ewing sarcoma, and correlation analysis miR-127-3p with P53, Ki-67 and EGFR molecules in tumor and the relationship between the clinicopathological parameters of patients. To provide experimental evidence for new therapeutic targets and biomarkers for Ewing sarcoma patients.In this study, Firstly, we will detecting differential expression of microRNAs in Ewing sarcoma which is associated with prognosis, and the correlation analysis and its clinical significance of miR-127-3p and P53, EGFR and Ki-67.Secondly, we will investigating the expression of EZH2 in Ewing sarcoma tissues by immunohistochemistry and analyzed its clinical significance; and we also followed-up the patients.[Materials and methods] A tissue microarray was constructed with a total of 81 cases diagnosis of Ewing sarcoma/primitive neuroectodermal tumor and other small round cell malignant tumors. Ewing sarcoma region 1(EWSR1) gene rearrangement was evaluated using fluorescence in situ hybridization (FISH). Respectively selected 3 cases of paraffin tissue samples which survival time less than one year and more than 3 years sent to the company to do microRNAs microarrays,and analysis differential expression of miRNA associated with prognosis. The expressions of EZH2, P53, Ki-67 and EGFR were determined using immunohistochemistry (IHC) in Ewing sarcoma.we analyze the correlation relationship between miR-127-3p or EZH2 and P53, Ki-67, EGFR expression and their prognosis. Cell function was evaluated by transfection, MTS, transwell assays.[Results] The first, according to microRNA microarrays,we test expression of hsa-miR-127-3p,hsa-miR-369-5p,hsa-miR-532-5p,hsa-miR-376c-3p and hsa-miR-497-5p.we find that the overall survival rate in miR-127-3p was remarkably higher than that in low group,and differences are significances (p=0.002), and the expression of miR-127-3p was correlated with survival time (p=0.002).Multivariate Cox regression analysis shown miR-127-3p expression was an independent risk marker of survival (HR=0.328,95%CI:0.102-1.055, P=0.016).In cellular level,miR-127-3p can inhibit the migration and invasion of the Ewing sarcoma cells, suggesting that miR- 127-3p play an role as tumor suppressor gene in the development of Ewing sarcoma. EZH2 and miR-127-3p have a negative relationship.The second, expression of EZH2 is closely related to lung metastasis (p=0.046), prognosis (p=0.004) and Ki-67(P=0.007) in Ewing’s sarcoma tissues. Kaplan-Meier analysis showed that the higher expression of EZH2 was identified in those patients with shorter survival time (P=0.006 and P=0.023). Cox multivariate analysis revealed that EZH2 was independent risk factors for overall survival in patients (HR=3.467, 95%CI=1.138-10.563, P=0.029). In cellular level, we found that after EZH2 interference significantly reduced the Ewing’s sarcoma cell migration, invasion and proliferation, suggesting that EZH2 play an oncogene role in Ewing’s sarcoma.[Conclusion] EZH2 and miR-127-3p could be used as a new prognosis biomarker and may be used as a molecular target for the treatment of Ewing sarcoma. At the same time, miR-127-3p can inhibit the occurrence and development of Ewing sarcoma, and it is found that EZH2 and miR-127-3p have a certain negative correlation.