The Role Of H3K9Methyltransferase SETDB1in Regulation Of Proliferation And Differentiationin Neuroblastoma

Neuroblastoma is a common childhood malignant tumor of neural crest origin,accounting for approximately6%-10%of pediatric cancers and15%of cancer-relateddeaths in children. Neuroblastoma is also a common malignant extracranial solid tumorof children, its annual incidence is about1/10million, just below the leukemia andcentral nervous system tumors. Neuroblastoma is an internal secretory nerve tumor,mainly originated from neural crest progenitor cells in the sympathetic nervous system.The most common location to find neuroblastoma is in the adrenal glands, but can alsooccur in the neck, chest, abdomen and other nerve tissue. Malignant degree ofneuroblastoma is high and its clinical manifestation includes high heterogeneity, easymetastasis, easy recurrence and poor prognosis, thus the treatment of neuroblastoma isdifficult. Only a small number of patients have had the tumor completely removed at anearly stage, the remaining cases prognosis is poorer. Even after combined treatments, itslong-term survival rate is still less than40%. Therefore, it is important to study thepathogenesis of neuroblastoma, explore the key targets for tumor cell proliferation anddifferentiation, and illustrate the mechanism of cell proliferation and differentiation,which can provide a new strategy for clinical treatment of neuroblastoma.Epigenetic regulation of tumor cell growth is one of the current research focuses, alot of experiments show that histone methylation plays a key role in the growth of thetumor. Histone methyltransferases have important theoretical and clinical value.SETDB1is a histone methyltransferase, mainly involved in H3K9me3methylation, theexisting research results show that SETDB1participate in mouse embryonicdevelopment, especially in nervous system development. In zebrafish experimentsSETDB1has been shown to promote the growth of melanoma, but the effect of SETDB1in neuroblastoma has not been well studied. This study based on methyltransferaseSETDB1and five kinds of neuroblastoma cells, using the existing clinicalneuroblastoma database, analyzing the relationship between the prognosis ofneuroblastoma and SETDB1. Weinvestigate the role of SETDB1in the cell proliferationand differentiation in neuroblastoma cells by knockdown or overexpression of SETDB1,which will provide theoretical basis for clinical treatment for neuroblastoma. The mainresearch results are as follows:①SETDB1expression in neuroblastoma is associated with poor prognosis In this study, we investigate the possibility of SETDB1as a prognostic marker inneuroblastoma.We had undertaken a microarray-based search using the online R2:microarray analysis and visualization platform. The database shows that high SETDB1expression is associated strongly with poor outcome, and vice versa, low SETDB1expression is correlated with good overall survival. Moreover, SETDB1expression isdifferent in various tumor stages, and increases significantly in stage4tumors. Asyounger patients at diagnosis are associated with good prognosis, we examined thecorrelation of SETDB1expression levels with patient diagnosed age by usinginformation available from the Versteeg database, and the result revealed that SETDB1expression level is significantly higher when patients diagnosis age are beyond18months, and consequently18months of diagnosis age is defined as the keydeterminants of risk nodes. We confirme that high N-MYC expression has a positivecorrelation with SETDB1expression. And the amplification of N-MYC is associatedclosely with the occurrence and recurrence of neuroblastoma. Because theneuroblastoma originated from the neural crest cells, which remind us that SETDB1may be involved in the regulation of oncogenes or tumor suppressor genes areexpressed in the process of embryonic development, make the control system of thesympathetic nervous system development happens disorder, prompting the formation ofneuroblastoma. Above all, this results suggest that SETDB1can be as an importantparameter in the risk stratification of patients with neuroblastoma.②Inhibition of SETDB1represses neuroblastoma cell growth and proliferationTo elucidate the contribution of SETDB1in neuroblastoma cell proliferation, weshow that this effects by knockdown and overexpress SETDB1. We undertookknock-down of SETDB1in BE-2-C and SHEP1cells by infection withlentivirus-expressing shRNA plasmids,and GFPsi as a negative control. We find cellmorphology changes significantlyand cell number reduces dramatically. And then weperformed CCK-8assay to detect the proliferation capabilities of the two groups.Theresults show that BE-2-C and SHEP1cells proliferation is suppressed completely afterknock-down of SETDB1. BrdU and ki67immunofluorescencestaining reveal that BrdUpositive rate and Ki67positive rate are reduce dramatically after SETDB1si. Further byusing flow cytometry instrument to detect the cell cycle, the results show thatSETDB1si induce cell cycle arrest in G1phase. Western blotting analysis showsthe G1phase of the specificity Cyclins and CDK2protein expression levels significantlylower.These results showed that SETDB1inhibition in neuroblastoma cells completely blocked cell proliferation, with cell arrested in the G1phase.Soft agar assaydemonstrate that the SETDB1si represses the neuroblastoma clone formation ability. Wefuther carried out xenograft study in NOD/SCID miceto examine the effect ofdownregulation of SETDB1on neuroblastoma, and we find neuroblastoma cells cannotgrow tumor in SCID mice after SETDB1si. To explore the role of SETDB1more, weperformed a recover experiment of overexpression SETDB1in cells which hadknockdown the SETDB1.The results show that after rescue the expression of SETDB1,the proliferation and growth of neuroblastoma cells, and its clone formation ability,tumorigenic potential has been restored. The results demonstrat that inhibition ofSETDB1decreases cell proliferation of neuroblastoma cells.③The histone H3K9methylation transferase SETDB1plays key role inneuroblastoma purine nucleotide synthesisTo investigate the molecular mechanism of SETDB1relates to cell proliferation,we perform an expressionmRNA microarray analysis of SETDB1si-cells, comparedwith control GFPsi, we find SETDB1is involved in the purine metabolism. Afterfurther analysis, the data shows that SETDB1si inhibition purinede novo synthesis keyenzyme gene expression significantly. Through the CCK-8experiment found that aftercompensate IMP, AMP, GMP in SETDB1si-cells, cells obtain part of growth ability.PRPS1is a key enzymes in Purine synthetic way, which catalyzed5-phosphate-ribose into5-phosphoribosyltransferases pyrophosphate (PRPP). Similar results arealso obtained with PRPS1interference, such as constraints on cell proliferation, cellclones diminished ability, etc. These results suggest that SETDB1si inhibiting thesynthesis of purine nucleotides.④The relationship between Histone H3K9methylation transferase SETDB1and neuroblastoma cell differentiationMany reports show that there is a small group neuroblastoma cells called cancerstem cells, which can cause the occurrence of tumor development and maintain thecharacteristics of the tumor, conferring cells with unlimited replicative potential, keepthe ability of self-renewal capacity and differentiation potential. In young patients, asmall number of patients can be converted to benign tumor naturally or spontaneousregression, some tumor even shrink or disappear at little or no treatment system. Astudy reported neuroblastoma induced by retinoic acid direction differentiation toneurons, expressed neuronal markers, such as (NEFL, NEFM, NEFH, MAP2, ENO2,DBH, etc.), can also be induced by BrdU to glial cell differentiation, expression of glial cell markers (GFAP, Vimentin, ALDH1L1, etc.). Bmi-1, MYCN gene,and Nestin inmaintaining neuroblastoma plays a very important role in maintain stemness. In thisstudy, we found that up-regulated SETDB1did not significantly increase theproliferation of neuroblastoma, but soft agar experiments shows obviously increase theself-renewal capacity of neuroblastoma. This suggests that higher SETDB1increasedneuroblastoma stemness. Through the analysis of expression profile data, we found thatthe SETDB1si caused maintainance of neuroblastoma stemness gene expressiondecreased, marker gene expression of neurons to decline, but the expression of glial cellmarkers to rise. Furthermore, by fluorescence quantitative PCR detection analysis, wediscovered that SETDB1si caused neuroblastoma stemness maintain gene MYCN,Bmi-1expression significantly decreased, neuronal markers such as NEFL, NEFM,NEFH, MAP2, ENO2and DBH expression quantity significantly reduced, but the glialcell marker GFAP expression, Vimentin, ALDH1L1volume increased. RestoringSETDB1expression, MYCN, Bmi-1expression also increased at the same time,neurons NEFL marker genes, NEFM, NEFH, MAP2, ENO2and DBH also recovered,but the glial cell marker GFAP, Vimentin, ALDH1L1significantly reduced. Theseresults suggest that SETDB1si can promote neuroblastoma to glial cell differentiation.All above results show that the SETDB1si reduces neuroblastoma cells growth andproliferation, and SETDB1si inhibits purine nucleotides de novo synthesis. SETDB1isclosely related to the stemness maintain, SETDB1si increases glial cell markerexpression. SETDB1plays an important role in neuroblastoma proliferation anddifferentiation, also include self-renewal ability, which providing theory basis for thefurther understanding of the pathogenesis of neuroblastoma and new therapeutic targets.