Topo??, H3K27me3 And JMJD3 Expressions,and Their Relevation With Neuronal Differentiation In Medulloblastoma

Medulloblastomas?MB?are a diverse set of cerebellar tumors,and are the most aggressive form of brain tumors occur in infants and young children.The annual incidence rate is 2.19⁶.60/10⁶.To date,a combination of surgery,radiation,and cytotoxic chemotherapy has been successful in eradicating the disease for a majority of patients,and current treatment protocols boast a 70%to 75%5 year overall survival.However,the outlook for patients with tumor dissemination or recurrence remains poor,with a 5 year survival rate of less than 40%.Moreover,medulloblastoma patients often experience treatment-related side effects,including long-term neurocognitive and neuroendocrine dysfunctions,and hearing loss,among others.Therefore,new approaches are needed to determine the mechanisms underlying medulloblastoma occurrence,to improve the survival rate,and to reduce the negative side effects of medulloblastoma treatment.Topoisomerase???Topo???,a protein specific expression in neuron,and is essential for cerebellum development.However,we current know little about its role in medulloblastomas.The jumonji domain containing protein 3?JMJD3?,an H3K27me3 specific demethylase,acting as a transcriptional activator,is also implicated in regulation of cerebellum development,epigenetically.However,there are still less studies regarding JMJD3 function in the formation of medulloblastomas.In this purpose,the present study is designed to evaluate Topo??,H3K27me3,MAP2 and JMJD3 expressions in medulloblastoma specimens obtained from clinics,and to assess their relevance with differentiated state of the tumor cells.Furthermore,we used13-cis Retinoic Acid?13-cis RA?to induce medulloblastoma cell model,Daoy cells to differentiate into neurons.Then,we examined the expressions of Topo??and JMJD3 in the cells underwent neuronal differen-tiation,and discussed their possible roles in use both for neurobiological research and for effective therapy of medolloblastoma in future.This study is composed of three parts as follows.Part one Topo??,H3K27me3 and JMJD3 expressions in medulloblastoma tissuesObjective:To evaluate Topo??,H3K27me3 and JMJD3 expressions,and the relevance with neuronal differentiation in medulloblastoma tissues.Methods:1 The clinical pathologic classification of medulloblastoma:11 cases of classic medulloblastoma,6 cases of desmoplastic medulloblastoma,and 7adjacent normal cerebellum tissues were determined by HE staining method.2 The protein expression levels of Topo??,H3K27me3,JMJD3 and MAP2 were detected by immunohistochemistry?IHC?with Streptavidin-Peroxidase?SP?in 11 cases of classic medulloblastoma,6 cases of desmo-plastic medulloblastoma,and 7 adjacent normal cerebellum tissues.3 The Gray value semi-quantitative detection was conducted by using the Motic Med 6.0 pathological graphic analysis system.4 The protein expression levels of Topo??,H3K27me3,MAP2 and JMJD3 were examined with Western blot in classic medulloblastoma,desmoplastic medulloblastoma,and adjacent normal cerebellum tissues.5 Statistical analysisThe results were expressed as mean±standard deviation?SD?and evaluated with SPSS 13.0 software by analysis of variance?One-Way ANOVA?.P<0.05 was considered statistically significant.Results:1 The positive expression rate of Topo??,JMJD3 and MAP2 in normal cerebellum tissues was higher than that in classic medulloblastoma.In desmoplastic medulloblastoma,the positive expression rate of Topo??at the intraocular regions was higher than that at internodular regions.The positive expression rate of H3K27me3 in normal cerebellum tissues was lower than that in classic medulloblastoma.In desmoplastic medulloblastoma,the positive expression rate of H3K27me3 at the intraocular regions was lower than that at internodular regions?P<0.05?.2 The gray value of Topo??,JMJD3 and MAP2 in normal cerebellum tissues was lower than that in classic medulloblastoma.In desmoplastic medulloblastoma,the positive expression rate of Topo??at the intraocular regions was lower than that at internodular regions.The positive expression rate of H3K27me3 in normal cerebellum tissues was higher than that in classic medulloblastoma.In desmoplastic medulloblastoma,the positive expression rate of H3K27me3 at the intraocular regions was higher than that at internodular regions?P<0.05?.3 The protein expression levels of Topo??,JMJD3 and MAP2 by Western blot in adjacent normal cerebellum tissues were higher than that in classic medulloblastoma and desmoplastic medulloblastoma.The protein expression levels of H3K27me3 by Western blot in adjacent normal cerebellum tissues were higher than that in classic medulloblastoma and desmoplastic medulloblastoma?P<0.05?.4 The fields with low expression of Topo??were coincided with the regions with high H3K27me3 or low JMJD3 expression in medulloblastoma tissues.Conclusions:1 The present study showed for the first time that high level of Topo??detected in adjacent normal cerebellum tissues,was shown to be consistance with the expressed pattern of MAP2,a maker of neuron,suggesting that Topo??is correlated with neuronal differentiation of cerebellum.2 Topo??displayed a low level in the tissue of classic medulloblastoma,and with high level at the intraocular regions of desmoplastic medullob-lastoma which is recognized as well differentiated type of medulloblastomas.These results suggested that Topo??might play some roles in occurrence of medulloblastoma,and that the low index of Topo??may indicate high malignant medulloblastomas.3 The epigenetic factors,H3K27me3 and JMJD3 were shown to be associated with medulloblastomas in this study.Their positive or negative relation with Topo??,suggested that the two factors may participate in the formation of medulloblastomas through regulation of Topo??gene expression.4 This study provided the basic materials for continuous our experiment by using larger numbers of medulloblastoma samples in futurePart two The effects of 13-cis RA on the growth and neuronal differentiation of Daoy cellsObjective:To study the effect of 13-cis RA on the growth characteristics and the neurons differentiation of Daoy cellsMethods:1 Cell culture:Daoy cells were cultured with the DMEM-H culture medium?10%fetal bovine serum?FBS?,penicillin 100 U/mL,streptomycin100?g/mL,4.5g/Liter Glucose?at 37?in humidified incubator with 5%CO₂.2 Cell growth assay:The log-phase Daoy cells were seeded in 96-well cell culture plates respectively with 1×10⁴cells per well.After adherence,cell was continually incubated with 13-cis RA for 5 days.Methyl thiazolyl tetrazolium?MTT?assay was used to detect the optical density?OD?of the cells at 490 nm wavelength everyday and cell growth curves were drawn.3 Cell cycle assay:The log-phase Daoy cells were seeded in 6-well cell culture plates respectively with 5×10⁴ cells per well.After adherence,cell was continually incubated with 13-cis RA for 5 days.The cell of 0 and 5 day were collected and were detected by flow cytometry.4 Cell apoptosis assay:Cells were collected on 0 and 5 days after 13-cis RA treament.The apoptosis rate was evaluated by flow cytometry.Nuclear staining was conducted on 0,1,3 and 5 days by DAPI and karyomorphology was observed.Western blot was used to detect the expression of Active caspase-3 protein.5 Identification of neuronal differentiation in each group:The cell numbers and neurite outgrowth were evaluated and analyzed.Differentiated cells percentages were calculated.To detect the expression of the neuronal marker MAP2 protein by Western blot.6 Statistical analysis:The results were expressed as mean±standard deviation?SD?and evaluated with SPSS 13.0 software by analysis of variance?One-Way ANOVA?.P<0.05 was considered statistically significant.Results:1 Cell proliferation tested by MTT assay.13-cis RA treatment with 10?mol/L and 20?mol/L showed no inhibition effects on the cells?P<0.05?.However,13-cis RA treatment with 0.1,1,5?mol/L showed no inhibition effects on the cells?P>0.05?.Cells treated with20?mol/L 13-cis RA were most obviously affected.The data indicated that13-cis RA could inhibit the cell proliferation in a time and dose dependent manner.2 Effects of 13-cis RA on cell cycle and apoptosis.Flow cytometry showed:compared with the control group,the number of G0/G1 phase cells was increased in the 13-cis RA treated group,and there was significant difference between the two groups?P<0.05?,compared with the control group,the apoptosis rate was no significant difference between the two groups?P>0.05?.Followed by DAPI staining,there were no nuclear fragmentation,apoptotic body and other apoptotic changes.The Active caspase-3 expression showed no significant difference between the two groups?P>0.05?.3 Morphological changes and percentage of differentiation of Daoy cells.The growth of Daoy cells treated with 10?mol/L 13-cis RA was significant inhibited?P<0.05?,while the growth of cells treated with0.1?mol/L,1?mol/L and 5?mol/L were not obviously affected by compared with the control group?P>0.05?.Those cells in 10?mol/L 13-cis RA exhibited a neuron-like phenotype with the longest neurites over that of cells in other groups?P<0.05?.4 Neuronal differentiation identified by neuronal markers MAP2.MAP2 is mainly express in the cell bodies and dendrites of neurons,and can be used as a molecule marker to identify neuronal differentiation.In this study,we found that the expression of MAP2 was significantly increased in the cells after 13-cis RA?10?mol/L?treatment for 3 and 5 days.There were significant difference in comparing with the control cells?P<0.05?.Conclusions:1 Daoy cells treated with 13-cis RA can be induced into neuronal differentiation.2 Daoy cells can be induced into neuronal differentiation after 13-cis RA?10?mol/L?treatment for 3 and 5 days.Based on this,the study chooses the conditions for subsequent research.3 It could be a novel strategy for driving cells differentiating into neurons by using the drugs like 13-cis RA in therapy of medulloblastomas in clinic.Part three Topo??,H3K27me3 and JMJD3 expressions in 13-cis RA-induced neuronal differentiation of Daoy cellsObjective:To study the possible roles of Topo??,H3K27me3 and JMJD3 in neuronal differentiation of Daoy cells induced by 13-cis RA.Methods:1 Cell culture:Daoy cells were cultured with the DMEM-H culture medium?10%fetal bovine serum?FBS?,penicillin 100 U/mL,streptomycin100?g/mL,4.5g/Liter Glucose?at 37?in humidified incubator with 5%CO₂.2 PCR?RT-PCR?analyzes the Topo??mRNA expression.3 Western blot was used to detect the expression of the Topo??,H3K27me3 and JMJD3 proteins.4 Topo??inhibitor ICRF-193 inhibited the13-cis RA-induced neuronal differentiation of Daoy cells:The cell numbers and neurite outgrowth were evaluated and analyzed.Differentiated cells percentages were calculated.Western blot was used to detect the expression of Topo??and MAP2 protein.5 Chromatin Immunoprecipitation,ChIP assay to examine the association of H3K27me3 and JMJD3 to Topo??gene promoter.6 Statistical analysisThe results were expressed as mean±standard deviation?SD?and evaluated with SPSS 13.0 software by analysis of variance?One-Way ANOVA?.P<0.05 was considered statistically significant.Results:1 RT-PCR detection of Topo??mRNA expression.The expression of Topo??mRNA increased significantly after 3 and 5days upon 13-cis RA treatment in contrast to the control cell?P<0.05?.2 The expression of Topo??with 13-cis RA treatment by Western blot.The expression of Topo??in Daoy cells was up-regulated after 13-cis RA treatment?10?mol/L?for 3 and 5 days.?P<0.05?.3 Topo??inhibitor ICRF-193 inhibited the 13-cis RA-induced neuronal differentiation of Daoy cells.After 5 days of 13-cis RA treatment,measurement of average neurite length of single cell,the results showed that there was a significant difference between the ICRF-193 group and the Con group?P<0.05?.The results of differentiated cells percentages showed that there was a significant difference between the ICRF-193 group and the Con group?P<0.05?.4 The expression of Topo??and MAP2 with ICRF-193 treatment by Western blot.The expression of Topo??and MAP2 protein by Western blot showed that there was a significant difference between the ICRF-193 group and the control group?P<0.05?.5 The expression of H3K27me3 and JMJD3 with 13-cis RA treatment by Western blot.The expression of H3K27me3 in Daoy cell was down-regulated after13-cis RA treatment?10?mol/L?after 3 and 5 days?P<0.05?.The expression of JMJD3 in Daoy cells was up-regulated after 13-cis RA treatment?10?mol/L?for 3 and 5 days?P<0.05?.6 13-cis RA induced neuronal differentiation through enhancing JMJD3and abate H3K27me3 in the Topo??gene promoter.ChIP assay showed that JMJD3 and H3K27me3 were more tightly associated with the Topo??gene promoter during 13-cis RA induction differentiation of Daoy cells.The expression level of JMJD3 binding in Topo??promoter region was significantly higher than that in control group?P<0.05?.The expression level of H3K27me3 binding in Topo??promoter region was significantly lower than that in control group?P<0.05?.Conclusions:1 13-cis RA induced neuronal differentiation of Daoy cells through up-regulation of Topo??was detected.13-cis RA decreased the expression of H3K27me3,while it promoted JMJD3 expression in differentiated cells.2 JMJD3 may positively regulation of Topo??gene expression during neuronal differentiation of medulloblastoma cells.3 13-cis RA can be effectively used to study on the differentiation of neurons,and to treat malignant tumors of the nervous system in the clinics.