| The development of the nervous system is a very complex process in mammalian ontogeny.Neuronal differentiation is very important for the formation of nervous system.In the process of neuronal differentiation,signal molecules are closely coordinated with cell proliferation,cell cycle and cell differentiation,and regulate the development and maturation of neural cells.It is important to investigate the molecular mechanism of neuronal differentiation in the development of nervous system,nerve regeneration,neurodegenerative diseases,and the development and treatment of brain tumors.Topoisomerase II?(Topo II?)is a key protein to promote neural development and neuronal differentiation.E2 transcription factor-1(E2F1)is an important transcription factor,which plays an important role in cell proliferation,cell apoptosis and cell differentiation.Cilostazol(CLZ)is the antiplatelet drugs,has a protective effect on neuronal damage.Recently,it has been reported that CLZ can interfere with the binding of E2F1 to its target gene and promote neuronal differentiation,but the exact mechanism is not clear.In this study,we used human neuroblastoma SH-SY5 Y cells into neuron differentiation model;through up-regulated the expression of E2F1,detected the expression of E2F1 and Topo II? in cell differentiation,and to explore the molecular mechanism of neuronal differentiation,and then provided some basis for research and clinical treatment of nervous system diseases.There are three parts in this study.Part 1 Effects of E2F1 on the differentiation of SH-SY5 Y cells induced by retinoic acidObjective: To study the effect of transcription factor E2F1 on the growth of SH-SY5 Y cells and the relationship between SH-SY5 Y cells and RA induced neuronal differentiation.Methods:1 Cell culture: SH-SY5 Y cells were cultured with the DMEM culture medium in humidified incubator with 5% CO2.2 Plasmid transfection.E2F1 overexpression plasmid was transfected into SH-SY5 Y cells by Lipofectamine 2000,and the transfection efficiency was observed by fluorescence microscope,qRT-PCR and Western blot.3 Induced cell differentiation: E2F1 overexpression plasmid was transfected into SH-SY5 Y cells,retinoic acid(RA)10 ?mol/L was induced to differentiation for 3 days.4 Cell growth assay: MTT was used to detect the cell growth index before and after transfection,and the growth curve was drawn.The morphology of cells was observed by inverted microscope before and after transfection.5 Cell cycle assay: the cells of each treatment group were collected and the percentage of G0/G1,S and G2/M cells were detected by flow cytometry.6 Identification of neuronal differentiation in each group: detected the neurite outgrowth;immunofluorescence and Western blot analyzed the expression of MAP2.7 Statistical methods: The results were expressed as mean±standard deviation(SD)and evaluated by analysis of variance(One-Way ANOVA).P<0.05 was considered statistically significant.Results:1 The SH-SY5 Y cells were adherent and clustered,their bodies were small and round.Most of them were irregular and with short neurites,few of them were spindle shape.2 Transfection was detected by fluorescence microscope,qRT-PCR and Western blot.Over expression of E2F1 plasmid with green fluorescence,this was transfected into SH-SY5 Y cells by Lipofectamine 2000.After transfection with E2F1 plasmid for 1 day,2 days,3 days,the green fluorescent protein was observed under inverted fluorescence microscope in order to judge the transfection efficiency above 80%.Total RNA and total protein were extracted from E2F1 after transfection.qRT-PCR and Western blot technology were used to detect the expression of mRNA and protein in the E2F1 overexpressing cells,which were significantly higher than those of the non transfected group and empty vector group,and there was significant difference in E2F1(P<0.05).The results showed that E2F1 overexpression plasmid was successfully transfected into SH-SY5 Y cells.3 Cell growth conditionsSH-SY5 Y cells transfected with E2F1 over expression plasmid,MTT results showed that: compared with the control group(Con),E2F1 overexpression group(over)cells grew fastest,and the two groups had significant difference(P<0.05).After transfection,adding 10 ?mol/L RA induced differentiation,induction group(Con+RA)cells' growth rate decreased significantly compared with the control group(Con),while the overexpression of E2F1 into RA group(over+RA),the growth rate is not significantly reduced,there was no significant difference compared with the control group.4 Morphological changes of SH-SY5 Y cells differentiationE2F1 overexpressing plasmid was transfected into SH-SY5 Y cells,and the cells were induced by adding or not adding 10 ?mol/L RA,and were observed under inverted microscope after 3 days: in the RA untreated groups,there was no significant difference between the overexpression group and the control group(Con);In the RA treatment group,compared with the non induction group,the number of cells in RA induced differentiation group decreased,the cell body aggregated and the neurite length increased obviously.Measurement of average neurite length of single cell,the results showed that there was a significant difference between the Con+RA group and the Con group(P<0.05),while the length of neurite outgrowth of SH-SY5 Y cells in over+RA group was not significant.5 Effects of transfection of E2F1 overexpressing plasmid on cell cycle distributionE2F1 overexpression plasmid was transfected into SH-SY5 Y cells,flow cytometry showed: compared with the control group(Con),the number of G0/G1 phase cells was decreased by 7.354% and S phase increased by 5.071% in the E2F1 over expression group(over),and there was significant difference between the two groups(P<0.05),while there was no significant difference between empty carrier group(overC)and control group(Con).After transfection,the cells were induced differentiation by RA for 3 days,the Con+RA group cells,G0/G1 phase increased obviously and increased 23.597% compared with the control group(Con),S phase cells decreased significantly,decreased by 28.410%,the result indicated that the occurrence of G0/G1 arrest(P<0.05).Compared with the Con group,the G0/G1 phase cells increased by 8.224% and the S phase decreased by 9.942%(P<0.05)in the over+RA group.These results suggested that RA could induce cell cycle arrest and overexpression of E2F1 could promote cell cycle progression.6 Neuronal differentiation identified by neuronal markersNeuronal differentiation markers,microtubule-associated protein 2(MAP2)were used to identify neuronal differentiation.Immunofluorescence staining showed that E2F1 overexpression group(over)was weaker than in control group(Con).After 10 mol/L RA treatment of SH-SY5 Y cells,the expression of MAP2 not only increased in the cytoplasm,but also increased significantly in the neurites,and there was a marked increase in Con+RA group compared with group Con cells.While the expression of MAP2 was lower in over+RA group,there is no significant difference compared with Con group.The expression of MAP2 was detected by Western blot,and the results were same with the results of immunofluorescence.Conclusions: SH-SY5 Y cells induced by 10 ?mol/L RA can make the cells break away from the cell cycle and differentiate into neurons;overexpression of E2F1 can promote the proliferation of SH-SY5 Y cells and decrease the differentiation of neurons;the results showed that the level of E2F1 in cells could affect the differentiation of neurons,and the specific mechanism needs further study.Part 2 E2F1 inhibits the differentiation of SH-SY5 Y cells into neurons by negatively regulating the expression of Topo II?Objective: To evaluate the effects of E2F1 and Topo II? expression on the differentiation of SH-SY5 Y cells into neurons.Methods:1 Cell culture: SH-SY5 Y cells were cultured with the DMEM culture medium in humidified incubator with 5% CO2.2 The effects of the expression of Topo II? after transfected the E2F1 overexpressing plasmid into cells by using immunofluorescence,qRT-PCR and Western blot.3 qRT-PCR and Western blot were used to detect the expression of p27,cyclinD1,CDK4,E2F1 mRNA and protein.4 Chromatin immunoprecipitation assay(ChIP)was used to detect the recruitment and binding of E2F1 in the promoter region of Topo II? gene.5 Statistical methods: with the first part.Results:1 The effect of transfection of E2F1 overexpressing plasmid on the expression of Topo II?Immunofluorescence staining showed that the expression of Topo II? was in the nucleus,and it was lower in E2F1 overexpression group(over)than that of the control group(Con),however,there was no significant difference between empty carry group(overC)and control group(Con).After 10 ?mol/L RA induced SH-SY5 Y cells,Con+RA,overC+RA group cells compared with Con cells,Topo II? expression was significantly increased,while it was lower in over+RA group than Con group.The results of qRT-PCR and Western blot were agreement with immunofluorescence results.2 qRT-PCR detected p27,cyclinD1,CDK4,E2F1 mRNA expressionCompared with the control group,the expression of p27 mRNA in E2F1 overexpressing group(over)decreased significantly(P<0.05),while the expression of cyclinD1,CDK4 and E2F1 mRNA increased obviously(P<0.05).There was no significant difference between the empty carry group(overC)and the control group(Con).After RA induced SH-SY5 Y cells,Con+RA,overC+RA group cells compared with Con cells,p27 mRNA expression was significantly increased(P<0.05),while it was lower in over+RA group than that in Con group(P<0.05).The expression of cyclin D1,CDK4 and E2F1 mRNA in Con+RA group was lower than that in Con group(P<0.05),but it was higher in over+RA group than that in control group(P<0.05).3 The expression of p27,cyclinD1,CDK4,p-Rb and E2F1 protein was detected by Western blotCompared with the control group,the expression of p27 protein in over group decreased significantly(P<0.05),while the expression of cyclinD1,CDK4,p-Rb and E2F1 protein increased obviously(P<0.05).There was no significant difference between overC group and Con group.After RA induced SH-SY5 Y cells,Con+RA,overC+RA group cells compared with Con cells,p27 protein expression was significantly increased(P<0.05),while it was decreased in over+RA group(P<0.05).The expression of cyclinD1,CDK4,p-Rb and E2F1 protein in Con+RA group was lower than that in Con group(P<0.05),but it was higher in over+RA group than that in control group(P<0.05).4 E2F1 negatively regulates the expression of Topo II? at transcriptional levelChIP assay showed that there was a binding site of E2F1 in the promoter region of Topo II?,the expression level of E2F1 binding in Topo II? promoter region in E2F1 over expression group was significantly higher than that in control group(P<0.05).After induction of RA,the specific binding of E2F1 in the Topo II? promoter region decreased gradually with the time of induction differentiation,the expression level of Con+RA group was lower than that of Con group(P<0.05),over+RA group was higher than Con group(P<0.05).The results indicated that the E2F1 binding in the Topo II? gene promoter region was inversely proportional to the cell differentiation;high expression of E2F1 inhibited the differentiation of SH-SY5 Y cells by inhibiting the expression of Topo II? in the process of RA induced cell differentiation.Conclusions: RA could decrease the expression of E2F1 and induce the differentiation of SH-SY5 Y cells,which was related to the up regulation of Topo II?;the increase of intracellular E2F1 level can promote the expression of cell cycle related proteins and inhibit neuronal differentiation;E2F1 can binding in the promoter of Topo II? gene and regulate the expression of Topo II? in the process of neuronal differentiation;high expression of E2F1 enhances its recruitment in the promoter region of Topo II? gene,inhibit the expression of Topo II?,and inhibit neuronal differentiation.Part 3 Cilostazol induced neuronal differentiation through E2F1/Topo II? pathwayObjective: To investigate the expression of Topo II? in CLZ induced SH-SY5 Y cells differentiation into neurons,and the mechanism of E2F1/Topo II? pathway on neuronal differentiation.Methods:1 Cell culture: SH-SY5 Y cells were cultured with the DMEM medium which supplemented with 10% fetal bovine serum,100 U/mL penicillin and 100 ug/mL phytomycin with the environment of 37 ?,5% CO2,fully humidified atmosphere.2 MTT was used to detecte the concentration of CLZ in SH-SY5 Y cells.3 Percentage of cell differentiation: the change of cell neurite length in different group were observed and measured in 0~5 days.Cells were considered differentiated when the total neurite length was longer than 100 ?m for each cell.Differentiated cells percentages were calculated.4 Morphological observation of cell differentiation: 30 ?mol/L CLZ treatments on human SH-SY5 Y cells for 3 days,observed the morphological changes of cells before and after CLZ treatment,and detected the differentiation.5 Identification of neuronal differentiation: detection of neuronal differentiation marker protein MAP2 by immunofluorescence and Western blot.6 The expression of Topo II? was detected by immunofluorescence,qRT-PCR and Western blot before and after treatment with CLZ.7 qRT-PCR and Western blot technology detection of p27,E2F1,PCNA expression in mRNA and protein levels.8 ChIP detected the degree of E2F1 binding to Topo II? and proliferating cell nuclear antigen(PCNA)gene promoter region.9 Statistical methods: with the first part.Results:1 MTT showed that different concentrations of CLZ(10,20,50,100,200 and 400 ?mol/L)could inhibit the proliferation of human SH-SY5 Y cells in vitro for 1,2,3,4,and 5 days.With the increase of CLZ concentration and the prolongation of culture time,the inhibition of cell proliferation was gradually increased,which was statistically significant compared with the control group(P<0.05).These results suggested that CLZ inhibited the proliferation of SH-SY5 Y cells in a concentration and time-dependent manner.Median inhibitory concentration(IC50)software was used to calculate CLZ on SH-SY5 Y cells.The concentrations for 1~5 days were respectively 167.472±6.289,76.334±2.549,36.629±3.148,31.382±2.148,28.146±1.485 ?mol/L.2 Percentage of cell differentiationCLZ induced cell differentiation into neurons,10,20,50 ?mol/L CLZ in the 1~3 day's cell differentiation gradually increased,reached the highest on the third day.On the base of the IC50 results of CLZ on SH-SY5 Y cells for 1~5 days which were calculated by MTT,and 30 ?mol/L CLZ was used to induce the cell differentiation for 3 days.3 Morphological changes of cell differentiationCompared with the control group,the cell proliferation rate of CLZ induced cells decreased,the number of cells declined,the synapses extended,and the number and length of neurites increased gradually.After third days of induction,the cells had a number of elongated synapses,and the length of neurite growth was obviously increased,which was morphological features of mature neurons.Compared with control cells,there was significant difference(P<0.05).4 Identification of neuronal differentiationDetection the expression of MAP2 by immunofluorescence,the result showed that: after 30 ?mol/L CLZ induced cell differentiation for 3 days,the length of neurite significantly increased,compared with the control group,there was significant difference(P<0.05).Western blot also showed that the expression of MAP2 protein was significantly higher than that of the control group(P<0.05).The results showed that 30 ?mol/L CLZ could induce SH-SY5 Y cells to differentiate into neurons.5 The expression of Topo II? after CLZ treatmentImmunofluorescence was used to detect the expression of Topo II?,the result showed that nuclear staining of CLZ induced group was significantly enhanced.Both qRT-PCR and Western blot showed that the expression of Topo II? was higher than that of the control group(P<0.05).6 The expression of p27,E2F1 and PCNAThe mRNA and protein levels of p27 in the CLZ induced group were higher than those in the control group(P<0.05),while the expression of E2F1 and PCNA were lower than in the control group(P<0.05).The results showed that 30 ?mol/L CLZ could induce cell cycle arrest and inhibit cell proliferation.7 CLZ affects the binding of E2F1 in the promoter region of Topo II? and PCNAChIP showed that the binding of E2F1 in the promoter region of Topo II? and PCNA were significantly inhibited,during CLZ induced SH-SY5 Y cells differentiation(P<0.05).The results showed that: E2F1/Topo II? pathway is involved in CLZ induced SH-SY5 Y cells differentiation,CLZ promotes the transcription and expression of Topo II?,inhibits cell proliferation and promotes cell differentiation.Conclusions: In this study,we used the target gene PCNA,which is controlled by E2F1 as a control,investigated the effects of CLZ on the expression of E2F1 and Topo II?.The results showed that CLZ can induce SH-SY5 Y cells to differentiate into neurons by inhibiting the binding of E2F1 and Topo II? gene;it is further proved that the E2F1/Topo II? signaling pathway is involved in neuronal differentiation.It provides a new molecular target for the research of neural development,the development of related drugs and the treatment of nervous system diseases. |
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