DAB2IP Expression And Its Regulatory Mechanism In Melanoma Stem Cells

Objective:Melanoma is the main cause of death of skin tumors,and the existence of cancer stem cells is one of the decisive factors causing tumorigenesis,drug resistance,recurrence and metastasis.DAB2IP is a member of Ras-GTPase activator protein family and plays an anti-cancer role in multiple cancers.However,the specific mechanism of DAB2IP in melanoma stem cells is unclear.Therefore,the aim of this study is to elucidate the expression of DAB2IP in melanoma stem cells and its regulatory mechanism.Methods:Melanoma stem cells B16-F10 and A375 were isolated and enriched by three-dimensional soft fibrin matrix.Real-time PCR and Western blot were used to detect the expression of DAB2IP and integrins.The interaction between DAB2IP,integrin and Sox-2 was studied by knock-down and over-expression techniques.The effect of DAB2IP on the metastasis ability of melanoma cells was analyzed by cell invasion and migration experiments.Results:Compared with two-dimensional culture(2D),three-dimensional culture(3D)of mouse-derived melanoma cells and human melanoma cells could form spherical clones for 5 consecutive days,and highly expressed tumor stem cell markers.At the same time,the DAB2IP expression was significantly down-regulated in 3D cells.Inhibition of DAB2IP expression promoted the formation of spherical clones and cell invasion and migration,while overexpression of DAB2IP inhibited the formation of spherical clones and cell invasion and migration.The results showed that the expression of integrin beta 3(ITGB3)increased significantly in 3D cells,while the expression of ITGAV/ITGB1/ITGB5 did not change significantly.Then,we inhibited the transcription and protein expression of DAB2IP by small interfering RNA(siRNA).The expression of ITGB3 and Sox-2 increased significantly,and the phosphorylated Akt was also increased.On the contrary,when ITGB3 siRNA was used to inhibit its transcription and protein expression level,the DAB2IP expression was increased while Sox-2 expression and the phosphorylated Akt decreased.Finally,the expression of ITGB3,Sox-2 and phosphorylated Akt were inhibited after DAB2IP overexpression.These results suggest that ITGB3 and DAB2IP may have a mutually regulating relationship.Conclusion:In melanoma stem cells,DAB2IP expression was down-regulated,and probably regulated by ITGB3,thus affecting the migration and invasion of melanoma cells.