Quantitative Nuclear Proteomics Identifies that miR-137-mediated EZH2 Reduction Regulates Resveratrol-induced Apoptosis of Neuroblastoma Cells*

Abstract

Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4′-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment. Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4′-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment. Neuroblastoma is a tumor derived from primitive cells of the sympathetic nervous system and is the most common solid tumor in childhood, accounting for 15% of pediatric cancer mortality (1Park J.R. Eggert A. Caron H. Neuroblastoma: biology, prognosis, and treatment.Hematol. Oncol. Clin. North Am. 2010; 24: 65-86Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 2Goldsby R.E. Matthay K.K. Neuroblastoma: evolving therapies for a disease with many faces.Paediatr. drugs. 2004; 6: 107-122Crossref PubMed Scopus (41) Google Scholar). A subset of neuroblastoma will undergo complete regression or differentiation, whereas others often end fatally despite recent intensive multimodal therapy. Around 50% of patients are currently classified as high-risk for disease relapse. The long-term survival rate of neuroblastoma patients is less than 40% (3De Bernardi B. Nicolas B. Boni L. Indolfi P. Carli M. Cordero Di Montezemolo L. Donfrancesco A. Pession A. Provenzi M. di Cataldo A. Rizzo A. Tonini G.P. Dallorso S. Conte M. Gambini C. Garaventa A. Bonetti F. Zanazzo A. D'Angelo P. Bruzzi P. Italian Co-Operative Group for N Disseminated neuroblastoma in children older than one year at diagnosis: comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group for Neuroblastoma.J. Clin. Oncol. 2003; 21: 1592-1601Crossref PubMed Scopus (155) Google Scholar, 4Tang S.Q. Huang D.S. Wang J.W. Zhang X.F. Liu L.Z. Yu F. Yang G. Treatment of high-risk neuroblastoma with intensive chemotherapy, autologous peripheral blood stem cell transplantation, and 13-cis-retinoic acid.Zhonghua er ke za zhi. 2004; 42: 486-489PubMed Google Scholar). Many features of neuroblastoma have been found to be associated with its high-risk clinical outcome, such as MYCN oncogene amplification (5Huang M. Weiss W.A. Neuroblastoma and MYCN.Cold Spring Harb. Perspect. Med. 2013; 3: a014415Crossref PubMed Scopus (373) Google Scholar), allelic loss of chromosome 1p or 11q (6Maris J.M. White P.S. Beltinger C.P. Sulman E.P. Castleberry R.P. Shuster J.J. Look A.T. Brodeur G.M. Significance of chromosome 1p loss of heterozygosity in neuroblastoma.Cancer Res. 1995; 55: 4664-4669PubMed Google Scholar), DNA ploidy (7Look A.T. Hayes F.A. Shuster J.J. Douglass E.C. Castleberry R.P. Bowman L.C. Smith E.I. Brodeur G.M. Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study.J. Clin. Oncol. 1991; 9: 581-591Crossref PubMed Scopus (524) Google Scholar), and overexpression of receptor tyrosine kinases TrkA and EPHB6 (8Nakagawara A. Arima-Nakagawara M. Scavarda N.J. Azar C.G. Cantor A.B. Brodeur G.M. Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma.N. Engl. J. Med. 1993; 328: 847-854Crossref PubMed Scopus (605) Google Scholar, 9Tang X.X. Zhao H. Robinson M.E. Cohen B. Cnaan A. London W. Cohn S.L. Cheung N.K. Brodeur G.M. Evans A.E. Ikegaki N. Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10936-10941Crossref PubMed Scopus (85) Google Scholar). Although more and more evidences have been shown to elucidate the neuroblastoma pathogenesis, the targeted and effective treatments are still in development. Heritable epigenetic mechanisms, including DNA methylation, histone modifications, nucleosome remodeling, and noncoding RNAs, play an essential role in the regulation of the mammalian genome complexity. Recent advances have shown that global epigenetic abnormalities occur in human cancer cells. Polycomb protein histone methyltransferase enhancer of zeste homolog 2 (EZH2), which is aberrantly overexpressed in multiple types of human tumors, including neuroblastoma, specifically catalyzes trimethylation of histone 3 on Lys 27 (H3K27me3), a well-known histone mark associated with gene silencing (10Alimova I. Venkataraman S. Harris P. Marquez V.E. Northcott P.A. Dubuc A. Taylor M.D. Foreman N.K. Vibhakar R. Targeting the enhancer of zeste homolog 2 in medulloblastoma.Int. J. Cancer. 2012; 131: 1800-1809Crossref PubMed Scopus (68) Google Scholar). In neuroblastoma, EZH2 represses tumor suppressors CLU, NGFR, RUNX3, and CASZ1, which contribute to the genesis and maintenance of the high-risk phenotype of neuroblastoma (11Wang C. Liu Z. Woo C.W. Li Z. Wang L. Wei J.S. Marquez V.E. Bates S.E. Jin Q. Khan J. Ge K. Thiele C.J. EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR.Cancer Res. 2012; 72: 315-324Crossref PubMed Scopus (141) Google Scholar). MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a post-transcriptional level by binding to the 3′-UTR of to or of of expression or role in a of including neuroblastoma F. Wang R. Wang Q. L. Wang S. Yang MicroRNAs in Neuroblastoma: with a Res. PubMed Scopus Google Scholar). In neuroblastoma, miR-137 is and as a tumor suppressor by K. A. A. P. J. K. J. F. A. Eggert A. A. as a tumor suppressor in neuroblastoma by J. Cancer. 2013; PubMed Scopus Google Scholar). represses the expression of EZH2 in stem the and Li Li Li L. N.J. Li W. Zhao Jin P. between and epigenetic regulation in 2010; PubMed Scopus Google Scholar). Targeting epigenetic a for (3, 5, 4′-trihydroxystilbene, is a naturally occurring present in and potential of the in PubMed Scopus Google Scholar). the of of its and in A. apoptosis in human cancer PubMed Scopus Google Scholar), Yu Wei J.S. Khan J. and proteomic to high-risk Res. 2010; 9: PubMed Scopus Google Scholar), Q. J. A. global in Res. PubMed Scopus Google Scholar), and A. M. C. L. G. M. as in induced Sci. 2003; 72: PubMed Scopus Google Scholar). In that RSV activity in the and of M. L. C.W. J.M. activity of a derived from PubMed Scopus Google Scholar). RSV been in clinical for human potential of the in PubMed Scopus Google Scholar). have shown that RSV cell apoptosis A. L. G. C. P. the cell at Res. PubMed Scopus Google Scholar, J.M. on cell and of apoptosis by resveratrol in human cancer cell Res. PubMed Scopus Google Scholar), and cell at the A. L. G. C. P. the cell at Res. PubMed Scopus Google through a of key of cell survival such as J.M. on cell and of apoptosis by resveratrol in human cancer cell Res. PubMed Scopus Google Scholar), M. of are associated with inhibition of of the Res. 2000; Google Scholar), M. A. N. C. J. Smith J. S. London A.B. Cohn S.L. M. G.M. G. M.D. in childhood neuroblastoma: clinical and of Natl. PubMed Scopus Google Scholar), and and tumor suppressor genes such as M. S. G. J. S. G. G. J. A. R. J. B. I. MYCN gene with MYCN and neuroblastoma: and clinical J. 2010; Full Text Full Text PDF PubMed Scopus Google and and homolog M. A. N. C. J. Smith J. S. London A.B. Cohn S.L. M. G.M. G. M.D. in childhood neuroblastoma: clinical and of Natl. PubMed Scopus Google Scholar). the are In to RSV clinical for cancer therapy, we the in tumor suppression in neuroblastoma cells. In study, we stable isotope labeling with amino acids in cell culture a and for protein at a global to in the expression of proteins in neuroblastoma cells S.E. M. PubMed Scopus Google Scholar). We found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of neuroblastoma cell line Neuro-2a (N-2a) cells treated with RSV. EZH2 decreased and led to decreased H3K27me3 a tumor suppressors of CLU and NGFR by EZH2 were RSV which were in the apoptosis and tumor we found that EZH2 expression was by which was up-regulated RSV treatment. Inhibition of miR-137 rescued the RSV-induced EZH2 reduction and cellular findings an epigenetic mechanism involving miR-137-mediated EZH2 reduction in RSV-induced apoptosis of neuroblastoma which would be a key therapeutic target in neuroblastoma treatment. The neuroblastoma cell line Neuro-2a (N-2a) and human neuroblastoma cell line were from of were in and at in a with The of RSV on the of N-2a cells was by were in and treated with RSV at and for We for we to and the at for The in cells was in the was at by The cell was by the apoptosis was by and with cells were with for at and by for at in cells were by The was a a and a We to the of 3 and its protein the of was by of the cells were by The results were by N-2a cells for were in of and or of and for of cell the cells were treated with RSV and cells were treated with the of as the were as J. K. M.E. J.J. and histone in human stem Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). cells were in the of and for on The cells and The were by at and for and the were by the were in the 2 and of the of and A at for The were with and and at and for to The protein was by a of and cells were at and protein of protein were The protein was by and by the was and in 50% with The proteins were reduced by with for at and with for in and the proteins were by at a at The were from with in 50% for 2 time, and in a were with to an were on a with a were of in at a rate of for of was The was with and Full were in the with at to a target of The most from the in the were by and time, with a target of We the at for the and for the We the and as as The was to and proteins J. I. M. N. M. M. A to the for quantitative PubMed Scopus Google Scholar, J. M. high and protein PubMed Scopus Google Scholar). were and was The were by a from the protein with and with of The were as the protein were and protein the was to the were to the was to and was with at amino acids were as The rate was to at and protein levels for protein protein at one or was analysis, were as a for The protein were from the of or to the protein with the of of and were with J. M. and a quantitative with 2012; Scopus Google Scholar). proteins a were The of and down-regulated proteins for was by 3 system P. M. F. J.M. A. a for in gene PubMed Scopus Google Scholar), in which the was for The with a were for were and three with in to the RNA to the The RNA was to a first was on the was in as an for the quantitative were in RNA for of and were and quantitative was by on the with was as an for the quantitative were in were in the of 2 and the of and A and at for were by at and for of protein were by and to a The were with in with for at with the at and the for at and were from histone and were from and H3K27me3 were from The of protein were by The protein was to expression were N-2a cells in were with EZH2 or with cells were and apoptosis and quantitative were as N-2a cells in were treated with DZNep for and were and apoptosis and quantitative were as cells were in a and with of miR-137 or miR-137 and of or from in were system activity was to with miR-137 and were as in were with or with cells were treated with RSV for cells were and apoptosis and quantitative were as N-2a cells in were with miR-137 or miR-137 with cells were and apoptosis and quantitative were as were at three were by and shown by the of were by and was to of RSV a of cancer cell growth and cellular apoptosis in and in in a of A. apoptosis in human cancer PubMed Scopus Google Scholar, Z. S. the expression of by its of and 3 and in the of cell J. PubMed Scopus Google Scholar, J. S. of cancer cell of A is a 6: PubMed Scopus Google Scholar), we the cell and cellular apoptosis in N-2a neuroblastoma cells to a of RSV of and the of RSV on N-2a cells we to the cell shown in the growth of N-2a cells was by RSV. the inhibition of RSV was in a and an 50% reduction in cell that N-2a cells and RSV results that RSV N-2a cell growth in a is with and including and and and and of PubMed Scopus Google Scholar). The which on the is an The of was by DNA with RSV treatment. the of N-2a cells treated with RSV at The nucleus was than nucleus and and at the the loss of of apoptosis in N-2a cells RSV we for The cells be by by The of cells RSV Treatment with RSV for 40% apoptosis in N-2a cells. the we the 3 and its by shown in RSV induced 3 and that with RSV in apoptosis by and and 3 in N-2a cells. that RSV N-2a cell growth and induced apoptosis significantly, we to the in protein was shown in N-2a cells were in of and or of and The cells were treated with RSV for and the cells were treated with as and of proteins of the and were by and from were on and The were by the with and proteins The from the was shown in In proteins were in and proteins were in at three and the between the we a between of proteins with a were to be 395 proteins were up-regulated and 302 proteins were down-regulated RSV treatment. shown in the for up-regulated proteins and the for down-regulated A of proteins be found in we the expression of proteins of N-2a we the proteins The proteins were shown in the of up-regulated proteins were in and down-regulated proteins in results that the up-regulated proteins were in regulation of and proteins were in DNA DNA regulation of histone and of cell The is of in cancer epigenetic are as for cancer J. Yang L. W. Liu Yu Q. of complex gene repression apoptosis in cancer 21: PubMed Scopus Google Scholar). In the the of EZH2 was down-regulated with a of RSV treatment. that the of epigenetic complex EZH2 was decreased to RSV treatment. was the first that we found be in the cellular apoptosis triggered by RSV in In to the quantitative the levels of the complex and and complex and were and protein levels were by in N-2a cells treated with RSV for the of and protein levels were down-regulated RSV whereas the levels were the level of H3K27me3 was reduced whereas expression was as the of the is a histone methyltransferase responsible for which is a epigenetic mark for tumor suppressor silencing (10Alimova I. Venkataraman S. Harris P. Marquez V.E. Northcott P.A. Dubuc A. Taylor M.D. Foreman N.K. Vibhakar R. Targeting the enhancer of zeste homolog 2 in medulloblastoma.Int. J. Cancer. 2012; 131: 1800-1809Crossref PubMed Scopus (68) Google Scholar). we EZH2 for we EZH2 protein level in N-2a cells with of RSV and by The that RSV reduced EZH2 protein level in a and EZH2 was down-regulated with RSV we the EZH2 protein level in N-2a cells treated with RSV for 2 and In line with EZH2 was decreased in a and as as 2 EZH2 was reduced In neuroblastoma, H3K27me3 a of tumor suppressors including CASZ1, CLU, RUNX3, and NGFR (11Wang C. Liu Z. Woo C.W. Li Z. Wang L. Wei J.S. Marquez V.E. Bates S.E. Jin Q. Khan J. Ge K. Thiele C.J. EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR.Cancer Res. 2012; 72: 315-324Crossref PubMed Scopus (141) Google Scholar). RSV-induced EZH2 reduction tumor suppressor we the levels of genes by shown in CLU and NGFR levels were up-regulated RSV whereas levels which be in tumor further that EZH2 was in apoptosis knockdown and of EZH2 were by of or DZNep treatment. was N-2a cells. the level and protein level decreased to and of that in the and H3K27me3 level was down-regulated EZH2 we 3 and shown in 3 was and was we a EZH2 to further the inhibition of EZH2 DZNep the level of EZH2 reduced EZH2 protein level and the associated H3K27me3 level in a and of 3 and were DZNep results that knockdown and of EZH2 induced apoptosis in N-2a cells. In N-2a EZH2 was the protein level decreased RSV that EZH2 protein level was reduced through a post-transcriptional mechanism A of including and were to EZH2 expression in cancer cells and stem cells Z. J. W. H. Z. Liu H. C. in cellular of stem cells from 2013; Scopus Google Scholar, J. binding of tumor suppressor to that of Res. 2013; PubMed Scopus Google Scholar, M. S. tyrosine and cell in to DNA 2013; PubMed Scopus Google Scholar, M. R. J.J. gene repression through is by of repression 2013; PubMed Scopus Google Scholar). the regulation mechanism of EZH2 reduction, we the levels of and in neuroblastoma N-2a cells and human neuroblastoma cells RSV treatment. shown in miR-137 was up-regulated RSV in N-2a cells and cells. miR-137 was in RSV-induced apoptosis through EZH2 we was to N-2a cells and cells RSV treatment. EZH2 level rescued the reduction of EZH2 protein level and H3K27me3 level associated with RSV and Inhibition of miR-137 rescued the cell loss 3 and the of tumor suppressor CLU and NGFR In in N-2a cells and rescued the cellular apoptosis associated with RSV treatment. results that miR-137 in cellular apoptosis through EZH2 N-2a cells with or EZH2 level was EZH2 level and H3K27me3 level were up-regulated and was in cell miR-137 inhibition and loss was by target to the 3′-UTR of the target gene to or that miR-137 target to EZH2 we 3′-UTR of we the activity of as on EZH2 3′-UTR in to A of that overexpression of miR-137 activity of EZH2 3′-UTR gene in of the EZH2 3′-UTR targeted by miR-137 in the gene led to complete of the of miR-137 we the of miR-137 on EZH2 shown in overexpression of miR-137 reduced EZH2 protein level to 40% in N-2a cells and in the levels were which the of EZH2 by induced 3 and in N-2a cells and cells The results that miR-137 was a epigenetic target for cancer therapy. EZH2 is high miR-137 is in a of neuroblastoma which be associated with a poor of we and to of survival on EZH2 and miR-137 expression The results that expression of EZH2 and expression of miR-137 were associated with a poor with of and we to the for EZH2 and miR-137 which were and Among EZH2 expression were associated with miR-137 expression whereas The between EZH2 and miR-137 expression was a naturally occurring found in to the of its and apoptosis in types of tumor including neuroblastoma, the most common extracranial solid cancer in less is the RSV the in we an quantitative to in proteins in neuroblastoma N-2a cells. We 395 up-regulated proteins and 302 down-regulated of proteins were associated with histone Among these, the histone methyltransferase EZH2 protein level was decreased in a and RNA level was RSV treatment. The tumor suppressors CLU and NGFR were to EZH2 reduction, which were in the tumor suppression of RSV. EZH2 knockdown by and by DZNep induced apoptosis in N-2a cells. EZH2 expression inhibition was by which was RSV treatment. Inhibition of miR-137 rescued the EZH2 reduction, EZH2 target gene CLU and NGFR and apoptosis phenotype triggered by RSV treatment. of of the epigenetic in cancer including DNA methylation, histone modifications, remodeling, and noncoding RNAs, specifically The abnormalities of epigenetic regulation are associated with tumor and with Targeting epigenetic regulation abnormalities a for cancer therapeutic The polycomb an activity in histone is overexpressed in human C. R. L. W. A. J.W. K. W.A. R. Li J.S. G. of tumor by 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. N. K. C. A. EZH2 overexpression in with cancer PubMed Scopus Google Scholar). EZH2 is in tumor and poor through and in cancer cells S. M. C. The polycomb protein EZH2 is in of PubMed Scopus Google Scholar, regulation of in role of the histone methyltransferase PubMed Scopus Google Scholar, M. The EZH2 polycomb or of Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Li Z. Z. M. P. Yu Q. regulation of target gene expression by EZH2 in Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. A. A. A. S. A. K. K. S. loss of in cell and 2012; 21: Full Text Full Text PDF PubMed Scopus Google Scholar). EZH2 was shown to be in neuroblastoma and was crucial to the genesis and maintenance of poor of neuroblastoma by epigenetic repression of multiple tumor suppressor genes of CASZ1, CLU, RUNX3, and NGFR (10Alimova I. Venkataraman S. Harris P. Marquez V.E. Northcott P.A. Dubuc A. Taylor M.D. Foreman N.K. Vibhakar R. Targeting the enhancer of zeste homolog 2 in medulloblastoma.Int. J. Cancer. 2012; 131: 1800-1809Crossref PubMed Scopus (68) Google Scholar, C. Liu Z. Woo C.W. Li Z. Wang L. Wei J.S. Marquez V.E. Bates S.E. Jin Q. Khan J. Ge K. Thiele C.J. EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR.Cancer Res. 2012; 72: 315-324Crossref PubMed Scopus (141) Google Scholar). Targeting EZH2 activity been as an for neuroblastoma cells (10Alimova I. Venkataraman S. Harris P. Marquez V.E. Northcott P.A. Dubuc A. Taylor M.D. Foreman N.K. Vibhakar R. Targeting the enhancer of zeste homolog 2 in medulloblastoma.Int. J. Cancer. 2012; 131: 1800-1809Crossref PubMed Scopus (68) Google Scholar, C. R. L. W. A. J.W. K. W.A. R. Li J.S. G. of tumor by 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. S. Liu Yu Q. The histone methyltransferase and cells in PubMed Scopus Google Scholar). In the study, we found that RSV decreased EZH2 which to reduced H3K27me3 the tumor suppressor CLU and NGFR by EZH2 were at level RSV treatment. findings for the first time, that EZH2 is a target for the of RSV in neuroblastoma cells. of expression or role in of are in tumor and through the expression of and tumor suppressor genes. recent that EZH2 expression was by and in cancer cells K. M. The enhancer of zeste homolog 2 (EZH2), a potential therapeutic is by in cancer Res. 2012; PubMed Scopus Google Scholar, W. A.B. J. M. S. B. Liu of homolog 2 is overexpressed in and is by and 2010; PubMed Scopus Google Scholar). miR-137-mediated EZH2 is in Li Li Li L. N.J. Li W. Zhao Jin P. between and epigenetic regulation in 2010; PubMed Scopus Google Scholar). that levels of miR-137 expression with a poor of neuroblastoma patients K. A. A. P. J. K. J. F. A. Eggert A. A. as a tumor suppressor in neuroblastoma by J. Cancer. 2013; PubMed Scopus Google Scholar). that EZH2 expression was by miR-137 in neuroblastoma which would contribute to tumor In our quantitative EZH2 as a in the RSV-induced apoptosis of neuroblastoma cells. We that the of EZH2 expression in tumor suppression of RSV. we that up-regulation of miR-137 was responsible for the repression of EZH2 our a involving miR-137 EZH2 reduction and tumor suppressor CLU and NGFR which to the cellular apoptosis of neuroblastoma cells triggered by RSV. findings our of the mechanism and therapeutic of RSV in neuroblastoma therapy. with enhancer of zeste homolog 2 resveratrol Neuro-2a stable isotope labeling with amino acids in cell culture polycomb complex 2 histone Lys 27 trimethylation A growth