| Objective: To study the effects of transcriptional regulation of Topo Ⅱβgene expression by sp1during the neuronal differentiation of SH-SY-5Y cells,and further to explore the possible mechanism of neuronal differentiationinduced by ATRA.Methodes:1Cell culture: SH-SY-5Y cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with10%fetal bovine serum (FBS),1%B27,penicillin (100U/ml) and streptomycin (100U/ml) at37°C in ahumidified atmosphere (95%air,5%CO2).2ATRA concentration chosen: SH-SY-5Ycells were incubated withdifferent concentration of ATRA(0,1,5,10,25,50,100μmol/L) in vitro.MTT assay were used to determine the influence of ATRA on the proliferationof SH-SY-5Ycells, the growth curve were drew and calculated the IC50of24h~120h.3Differentiated cells percentage of SH-SY-5Y cells:SH-SY-5Ycells wereincubated with different concentration of ATRA(1,5,10,25,50μmol/L) invitr. To demonstrate that SH-SY5Y cell differentiation induced by ATRA,200cells were observed using inverted microscope at different times (6h,24h,72h,120h) fou each group. We tested the cell numbers, cell body diameter, andextension of neuritis outgrowth for determaining mature neurons. The resultsbe analyzed with software Image-Pro Plus6.0and calculated the differentiatedcells percentage of SH-SY-5Y cells.4Morphological properties of SH-SY-5Y cells induced by ATRA:①Control group: Change the medium with DMEM medium containing10%FBS,1%B27, penicillin (100U/ml) and streptomycin (100U/ml), and added the10μmol/L DMSO into the medium.②ATRA group: Change the medium with DMEM medium containing3%FBS,1%B27, penicillin (100U/ml) andstreptomycin (100U/ml), and added the10μmol/L ATRA into the medium. Todemonstrate SH-SY5Y cells differentiation with ATRA,200cells wereobserved by inverted microscope at different times (6h,24h,72h,120h) foreach group. We tested that the cell numbers, cell body diameter, and extensionof neuritis outgrowth with morphological similarity to mature neurons. Theresults be analyzed with software Image-Pro Plus6.0.5Cell cycle revealed by Flow Cytometry:①ATRA group: change themedium with DMEM medium containing3%FBS,1%B27, penicillin(100U/ml) and streptomycin (100U/ml), and added the10μmol/L ATRA intothe medium.②Control group:SH-SY-5Y cells were cultured with DMEMmedium containing10%FBS,1%B27, penicillin (100U/ml) and streptomycin(100U/ml), and added the10μmol/L DMSO into the medium.After culturedfor three days, cells were harvested and cell cycle were analyzed with FlowCytometry.6RT-PCR analysis: The expression of Sp1, TopoⅡ α, TopoⅡ β mRNAwere detected in the SH-SY-5Ycells induced by10μmol/L ATRA.7Western blotting analysis: The expression of MAP2,Sp1, TopoⅡα,TopoⅡβ proteins were detected in the SH-SY-5Ycells induced by10μmol/LATRA.8Chromatin immunoprecipitation assays: To assess the role of Sp1transcriptional regulation binding to the GC elements of the promoter ofTopoⅡ β, we analyzed their binding ability to the GC boxes by ChIPexperiment.Results:1The cultured SH-SY5Y cells adhered on the bottom of the flask, theirbodies shown irregular polygon. Most of them have many short synapses.2Cell inhibition effect measured by MTT assay: The proliferation ofSH-SY5Y could be inhibited by different concentrations of ATRA (1,5,10,25,50,100μmol/L). In the concentrations range of1~25μmol/L, ATRA couldinhibit the proliferation of SH-SY5Y in a dose and time dependent manner. Cell numbers were decreased significantly when cells were treated with50μmol/L and100μmol/L. With the application of IC50calulation softwarewe get half of the inhibition concentration of SH-SY5Y cells to ATRA for24h~120h (24h:34.259±2.183μmol/L;48h:19.621±1.540μmol/L;72h:10.330±0.942μmol/L;96h:7.876±0.625μmol/L;120h:5.706±0.490μmol/L).3In the concentrations range of5~25μmol/L, with the increasing time oftreatment(6h~72h), differentiated cell percentage of SH-SY-5Y cells gothigher apparentely. Differentiated cell percentage of SH-SY-5Y cells treatedfor72h with10μmol/L ATRA was78.540±2.037%. Results showed thatSH-SY5Y cells treated for72h with10μmol/L ATRA was considered asbest-inducing condition and proceed with the following experimental study.4Morphology of SH-SY-5Y cells cultured with and without10μM ATRA:Cells were induced by10μmol ATRA on the6h, cell numbers and neuritelength was no significant change compared with control groups. On the72h,the cells exhibited a neuron-like phenotype with neuritis extending more thantwice the length of cell body when cells were induced by10μmol/L ATRA,while the undifferentiated cells maintained relatively short neuritis.5Cell cycle distribution revealed by Flow Cytometry: Compared with thecontrol group, the percentage of G0/G1cells increased25.2%, while thepercentage of G2/M phase cells decreased24.7%, the percentage of PIincreased25.2%(P<0.05). Results suggested that the cell cycle of cells afterinduced with10μmol ATRA were arrested at G0/G1phase, and the growth ofcells were inhibited.6RT-PCR analysis of expression of Sp1, Topo Ⅱα, Topo Ⅱβ mRNA inSH-SY5Y cells:The Expression of TopoⅡα mRNA was down-regulatedwhen cells treated with ATRA; The Expression of Sp1and TopoⅡβ mRNAwas up-regulated when cells treated with ATRA, but the mRNA expression ofTopoⅡα,TopoⅡβ, and Sp1was down-regulated when the cells treated withATRA containing of0.1μmol/L MithramycinA.7Western analysis of expression of MAP2, Sp1, Topo Ⅱα,Topo Ⅱβ proteinin SH-SY5Y cells: The Expression of TopoⅡα protein was down-regulated when cells treated with ATRA; The Expression of Sp1and TopoⅡβ proteinwas up-regulated when cells treated with ATRA, but the protein expression ofTopoⅡα,TopoⅡβ, and Sp1was down-regulated when cells treated withATRA containing of0.1μmol/L MithramycinA.8Chromatin immunoprecipitation assays: Results showed that Sp1occupancy was elevated in cells treated with10μmol/L ATRA compared toinput, and was reduced in cells treated with10μmol/L ATRA containing of0.1μmol/L MithramycinA compared to input. Results Sp1protein could couldup-regulated the expression of gene TopoⅡβ.Conclusions:1Results showed that the SH-SY5Y cells could be induced to neuronalwith ATRA. Differentiated cell percentage of SH-SY-5Y cells got higherapparentely when cells were induced with5,10,25μmol/L ATRA for6h~72h,and the differentiated cell percentage was the highest when cells were inducedwith10μmol/L ATRA for72h.2The mRNA and protein expression of TopoⅡα was down-regulated whencells were differentiated into neurons induced by ATRA. TopoⅡα wasdown-regulated in differentiated cells made a positive correlation to theinhibition the growth of cells and made a negative correlation to the cells ofdifferentiated into neurons.3The mRNA and protein expression of Sp1and TopoⅡβ was up-regulatedwhen cells were differentiated into neurons induced by ATRA, and the mRNAand protein expression of Sp1and TopoⅡβ was down-regulated when cellswere treated with ATRA containing of0.1μmol/L MithramycinA. Sp1andTopoⅡβ was up-regulated in differentiated cells made a positive correlation tothe cells of differentiated into neurons.4Sp1could up-regulated the expression of TopoⅡ β by Sp1binding to theGC elements of the promoter when cells were differentiating into neuronsinduced by ATRA. |
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