The Role Of Hoxc9 In Retinoic Acid-induced Differentiation Of Neuroblastoma Cells

PART 1 The molecular mechanisms of retinoic acid-induced differentiation of neuroblastoma cellsObjective To investigate the molecular mechanisms of retinoic acid-induced differentiation of neuroblastoma cellsMethods Retinoic acid was used to induce the differentiation of neuroblastoma cells. The cell cycle of cells was measured by flow cytometry after PI staining. Real time-PCR and western blotting were used to detect the expression of cyclins (cyclinA, cyclinB1, cyclinD1, cyclinE1, and cyclinE2) and CDKs (CDK1, CDK2, CDK4, and CDK6). The half-life of cyclins was assessed by western blotting and Image J software after the biosynthesis of cyclins was inhibited by protein synthesis inhibitor cycloheximide.Results RA induced differentiation and G1 arrest of neuroblastoma BE (2)-C cells. The levels of cyclinA, cyclinB1, cyclinE2 and CDK1 declined substantially with time following RA treatment. The half-life of cyclins (cyclinA, cyclinB1, and cyclinE2) was not apparently affected by RA treatment.Conclusions Retinoic acid induces growth arrest and differentiation of neuroblastoma BE (2)-C cells by downregulating the expression of cyclins (cyclinA, cyclinB1, and cyclinE2) and CDK1.PART 2 The role of Hoxc9 in retinoic acid-induced differentiation of neuroblastoma cellsSection 1 Upregulation of Hoxc9 expression in RA-induced differentiation of neuroblastoma cellsObjective To investigate the effect of RA on genes expression of neuroblastoma cells. Methods Microarray was used to detect the changes of cDNA level in BE (2)-C cells treated with RA. Real time PCR and Western blotting were used to confirm the result of microarray.Results Microarray showed that the expression of certain Hox genes were upregulated in BE (2)-C cells after exposure to RA. Real time PCR and Western blotting showed the mRNA and protein level of Hoxc9 increased substantially with time.Conclusions Hoxc9 may have a role in RA-induced differentiation of neuroblastoma cells.Section 2 Ectopic expression of Hoxc9 inhibites growth of neuroblastoma cellsObjective To investigate the effect of Hoxc9 overexpression in neuroblastoma cells.Methods After generating BE (2)-C-derived cells with inducible expression of Hoxc9 in the absence of doxycycline with the Retro-X Tet-Off advanced inducible gene expression system, MTT assay, Ki67 staining, flow cytometry, senescence assay, soft agar and xenograft assay were used to investigate the functional consequence of Hoxc9 upregulation in neuroblastoma cells. Real time PCR and western blotting were used to detect the molecular basis of Hoxc9 upregulation in neuroblastoma.Results Ectopic expression of Hoxc9 markedly inhibited the growth of neuroblastoma cells in culture, soft agar and immunodeficient mice. Hoxc9 overexpression also resulted in a marked reduction in levels of cyclinA, B1, E2 and CDK1 in BE (2)-C cells.Conclusions Ectopic expression of Hoxc9 inhibited the growth of neuroblastoma cells. Section 3 Hoxc9 is the key mediator of RA-induced differentiation of neuroblastoma cellsObjective To investigate the effect of Hoxc9 in RA-induced differentiation of neuroblastoma cells.Methods After downregulation of Hoxc9 in neuroblastoma cells with lentiviruses, we detected the changes of neuroblastoma cells treated with RA by trypan blue exclusion assay and Ki67 staining. The expression of cyclins (cyclinA, cyclinB1, cyclinD1, and cyclinE2) were assessed by Western blotting.Results Abrogation of Hoxc9 attenuated the RA-induced differentiation of neuroblastoma cells. RA-induced downregulation of cyclinA, cyclinB1, and cyclinE2 was also attenuated after inhibiting the downregulation of Hoxc9. Conclusions Hoxc9 is required for RA-induced G1 arrest and differentiation of NB cells.PART 3 The role of Hoxc9 in neuroblastoma cell cycle controlObjective To investigate the role of Hoxc9 in neuroblastoma cell cycle control.Methods After elimination of Hoxc9 and/or cyclin B1 expression by RNA interference, we investigated the morphologic changes of NB cell lines with microscope. Flow cytometry and immunofluorescence were used to detect cell cycle. The changes of the protein levels were detected by western blotting.Results Downregulation of Hoxc9 resulted in a dramatic increase in the number of cells that appeared round and refractile. These cells apparently arrested in M phase as indicated by FACS analysis and by their expression of pHH3, a marker for mitotic cells. The expression of cyclinB increased in Hoxc9 knockdown cells. RNAi-mediated downregulation of cyclin B1 was able to rescue the phenotype of Hoxc9 knockdown in NB cells.Conclusions Hoxc9 control the divition of NB cells by regulating cyclinB1 transcription.