The Role And Mechanism Of Histone Lysine Methyltransferase SETDB1 And Its Down Effector In Neuroblastoma Cell Proliferation And Differentiation

Neuroblastoma is one of the most common solid tumors with high malignant degree in children. Its clinical feature contains poor prognosis, easily metastasis and recurrence, and with high heterogeneity. And neuroblastoma has the characteristics of self-regression and differentiation in vitro, which makes it a good tumor differentiation model to explain the mechanism of neuroblastoma proliferation and differentiation, and can provide new strategy for the clinical treatment. Epigenetic regulation of cancer cell is a hotspot research; a lot of experiments show that histone methylation plays a key role in the growth of the tumor. Histone methyltransferases have important theoretical and clinical value. In recent years, it was found that aberrant methylation of histone modifications often made changes in gene expression, and become one reason for tumorigenesis. Study found that histone methyltransferase SETDB1, which catalyses H3K9 tri-methylation, is highly expressed in many types cancers, including small and non-small cell lung cancer, glioma, and prostate cancer. Downregulating its expression may inhibit tumor cell proliferation and differentiation, also have an effect on cell metastasis and invasion.We investigate the function of SETDB1 in cell proliferation and differentiation in neuroblastoma, and explore its mechanism. which could expand our horizons for clinical treatment. Our research reveals the vital function of SETDB1 and its downstream effector PRPS1 in proliferation and differentiation processes of neuroblastoma.The main research results are as follows: 1. Histone methyltransferase SETDB1 regulation neuroblastoma cell growth and differentiationFirstly, we downregulated the expression of SETDB1 using shRNA in BE-2-C and SHEP1 cells, GFPsi is as a negative control. Through performing CCK8 assays, and soft agar assays, we found that cell growth, self-renewal are suppressed significantly, and the tumorigenesis is also inhibited in vivo after SETDB1 si. And through the analysis of expression profile data, qRT-PCR(Quantitative Real-time PCR) assay, and western blot assay, we also found the SETDB1 si declined “stemness” gene expression, and also inhabited neurons marker gene expression, and increased glial cell marker gene expression. This data implies the SETDB1 si can promote neuroblastoma to differentiate to glial cell. 2. SETDB1 can regulate the expression of the rate-limiting enzyme of purine nucleotide synthesis PRPS1, and PRPS1 plays a key role in cell proliferation and growthExpression spectrum analysis of SETDB1si-cells demonstrated that SETDB1 involves in the purine metabolism, data also shows that purine key enzyme PRPS1 gene expression significantly declined in SETDB1 si, and we confirmed the result by qRT-PCR assay, western blot assay. And ChIP-qPCR assay showed that SETDB1 is bingding with the promoter of PRPS1, suggested that SETDB1 can regulate the expression of PRPS1. Then we analyzed PRPS1 from the online R2 database and found PRPS1 expression in neuroblastoma is associated with poor prognosis. Next we downregulated the expression of PRPS1 using shRNA in BE-2-C and SHEP1 cells, through performing CCK8 assays, Brd U staining and soft agar assays, we found that cell growth, cell proliferation and self-renewal are suppressed significantly, and flow cytometric analysis of PRPS1 si cell cycle found that cell cycle arrest in G1 phase, moreover, the tumorigenesis is also inhibited in vivo after SETDB1 si. These results are similar with SETDB1 interference. Our research shows that SETDB1 si inhibiting PRPS1 expression, and both of them play key roles in neuroblastoma growth and proliferation. 3. Histone methyltransferase SETDB1 is associated with N-MYCTo explore the reason of SETDB1 regulated cell differentiation, we analyzed the correlation of SETDB1 expression levels with oncogene N-MYC expression levels in online database, and confirmed that high SETDB1 expression along with N-MYC expression. N-MYC is considered the hallmark of neuroblastoma. It affects neuroblastoma cell self-renewal and differentiation, and have an important impact on the development of neuroblastoma. Through the analysis of expression profile data, qRT-PCR assay, and western blot assay, we discovered that SETDB1 si caused N-MYC expression decreased, and by MTT assays and BrdU staining assay, we found upregulated N-MYC expression after SETDB1 si in SHEP1, result shows overexpress N-MYC did significantly increase the proliferation of SETDB1 si neuroblastoma cells. To explain the relationship between SETDB1 and N-MYC, we performed ChIP-qPCR assay, and discovered that SETDB1 and H3K9me3 directly binds with N-MYC upstream promoter region-2885~-2723 bp, suggest that SETDB1 directly regulated N-MYC.Above results show that the SETDB1 si reduces the neuroblastoma cell proliferation and differentiation. And SETDB1 si regulates the expression of PRPS1, which is a key enzyme in purine nucleotides de novo synthesis, and PRPS1 is also involving in neuroblastoma cell proliferation. And SETDB1 is closely related to proto-oncogene N-MYC, and further affects the proliferation and differentiation of neuroblastoma. Our research provides new view of novel therapeutic targets of neuroblastoma. Also offer basic theory for the pathogenesis research of neuroblastoma.