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The Effect Of HOXA9 And HOXB9 Gene Expression In Cell Cycle Control And Differentiation Of Neuroblastoma Cells

Posted on:2018-01-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:W Y TaoFull Text:PDF
GTID:1314330515496280Subject:Neurology major
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Part1 The effect of HOXA9 and HOXB9 gene expression in neuroblastoma cell cycle controlObjective:To investigate the effect of HOXA9 and HOXB9 gene expression in cell cycle control of the human neuroblastoma cell lines BE(2)-C.Methods:The Retro-X Tet-Off Advanced Inducible Gene Expression System(Clontech)was used to generate BE(2)-C derived cell lines with inducible expression of individual HOXA9 and HOXB9 genes in the absence of doxycycline.Cells were examined and phase contrast images captured daily using inverted phase contrast microscope and software.For testing the effect of HOXA9 and HOXB9 to cell cycle,cells were examined by PI staining flow cytometry and the percentage of cell cycle phase was analyzed by software.For detecting the protein levels of cell cycle regulatory proteins(CyclinA2,CyclinB1 and CDk1)were examined by western blotting.All these tests were carried out in the absence of doxycycline and have time comparability for 3,6 and 9 days.Results:(1)The expression of HOXA9 and HOXB9 were not induced in BE(2)-C derived cells in the presence of doxycycline with the Retro-X Tet-Off advanced inducible gene expression system.Western blotting showed that the protein level of HOXA9 and HOXB9 began to be expressed in BE(2)-C cells cultured in the absence of doxycycline for 3 days and increased substantially with time.(2)With the high HOXA9 and HOXB9 expression in the human neuroblastoma cell lines BE(2)-C cultured in the absence of doxycycline,their growth was significantly slow,their density was obviously reduced,and their morphological changes were observed.(3)For testing the effect of high HOXA9 and HOXB9 expression to cell cycle,cells were examined by PI staining flow cytometry.With the high HOXA9 expression in the human neuroblastoma cell lines BE(2)-C cultured in the absence of doxycycline with the Retro-X Tet-Off advanced inducible gene expression system,cells in the percentage of Gl phase were increased from 68.7%in the control group to 85.8%(p<0.05).With the high HOXB9 expression in the human neuroblastoma cell lines BE(2)-C cultured in the absence of doxycycline with the same system,cells in the percentage of Gl phase were increased from 73.5%in the control group to 90.9%(p<0.05).(4)By using western blotting,the expression of HOXA9 and HOXB9 was increased substantially with time in BE(2)-C cells cultured in the absence of doxycycline.The expression level of HOXA9 and HOXB9 peaked at 9 days in BE(2)-C cells cultured in the absence of doxycycline,meanwhile,the expression of cell cycle regulatory proteins(CyclinA2,CyclinB1 and CDk1)was discreased substantially with time.Conclusions:(1)Tet-off inducible gene expression system was an ideal tool system for studying the function of HOX gene family in the regulation of cell fate,which provided a controllable security approach and research tool for tumor gene therapy.(2)Through downregulation of cell cycle regulatory proteins(CyclinA2,CyclinB1 and CDkl)in BE(2)-C,the high expression of HOXA9 and HOXB9 induced G1 arrest and thus inhibited the proliferation of tumor cells.(3)Regulation on cell cycle in human neuroblastoma cell lines BE(2)-C,HOXA9 and HOXB9 had similar function and was as a tumor suppressor in neuroblastoma.Part 2 The role of high HOXA9 and HOXB9 gene expression in differentiation of neuroblastoma cellsObjective:To investigate the role of high HOXA9 and HOXB9 expression in differentiation of the human neuroblastoma cell lines BE(2)-C.Methods:The Retro-X Tet-Off Advanced Inducible Gene Expression System(Clontech)was used to generate BE(2)-C derived cell lines with inducible expression of individual HOXA9 and HOXB9 genes in the absence of doxycycline.Cells were observed by inverted phase contrast microscope.Cell morphological changes and the expression of peripherin,vimentin and NEFM were examined by immunofluorescence staining,and the expression of PHOX2B and NEFM were also examined by Western blotting.Results:(1)With the high HOXA9 and HOXB9 expression in the human neuroblastoma cell lines BE(2)-C cultured in the absence of doxycycline,cell morphological changes were observed by inverted phase contrast microscope.The BE(2)-C bodies became thinner and sprouted long protrusions,similar to the morphological changes of neuron.Meanwhile,the cells became smaller and their growth became slow.(2)After the high expression of HOXA9 and HOXB9 in BE(2)-C cells cultured in the absence of doxycycline,the expression of KI67 cells was decreased and their cell morphologies were also changed,meanwhile,the expression of peripherin and NEFM were increased,and the expression of NEFM was reduced by immunofluorescence staining.(3)After the high expression of HOXA9 and HOXB9 in BE(2)-C cells cultured in the absence of doxycycline,the expression level of PHOX2B as an undifferentiated marker in neuroblastoma was significantly decreased,while the expression of NEFM was up-regulated by Western blotting.Conclusions:(1)The expression of cell proliferation factor Ki67 was downregulated,indicating that the high expression of HOXA9 and HOXB9 inhibited tumor cell growth.(2)The high HOXA9 and HOXB9 expression resulted in the high expression of peripherin,the low expression of PHOX2B,and the up-regulation of of NEFM in BE(2)-C cells by immunofluorescence staining,suggesting that the BE(2)-C cells would differentiate into N cells and further induce differentiation into neurons.(3)Regulation on cell growth and differentiation in human neuroblastoma cell lines BE(2)-C,HOXA9 and HOXB9 had similar function and act as a tumor suppressor in neuroblastoma.
Keywords/Search Tags:HOXA9, HOXB9, Neuroblastoma, Cell cycle, Differentiation
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