Mechanism of SPHK1 Regulation of M2 Macrophage Polarization on Epithelial Mesenchymal Transformation of Bladder Cancer Cells
Objective:To investigate the regulation of sphingosine kinascs-1(SPHK1)on M2 macro-phage polarization and its mechanism on epithelial mesenchymal transition(EMT)in bladder cancer cells.Methods:Raw264.7 macrophages were cultured using the primary culture method.The cells were divided in-to the following groups:Control-NC(empty SPHK1 plasmid-transfected Raw264.7 cells),LV-SPHK1(SPHK1 overexpression plasmid-transfected Raw264.7 cells),and si-SPHK1(SPHK1 knockdown plasmid-transfected Raw264.7 cells).Western blot was used to detect the expression of SPHK1 and M2 macrophage markers(CD206 and ArgI)proteins in each group.Immunofluorescence was used to detect the fluorescence intensity of M2 macrophage markers(CD206 and ArgI)proteins.After co-culturing the macrophages of each group with bladder cancer T24 cells for 24 hours,the MTT method was used to detect the proliferation activity of T24 cells in each group.The scratch and Transwell assays were used to detect the migration and invasion a-bilities of T24 cells in each group.Western blot was used to detect the expression levels of proliferation-and invasion-related proteins(Survivin,MMP-2,and MMP-9)and EMT-related proteins(E-Cadherin,Vim-entin,and N-Cadherin)in each group.Results:Compared with the Control-NC group,the SPHK1 protein expression was significantly increased in the LV-SPHK1 group and significantly decreased in the si-SPHK1 group(P<0.05).The overexpression of SPHK1 upregulated the expression and fluorescence intensity of M2 macrophage markers CD206 and ArgI proteins(P<0.05).The knockdown of SPHK1 downregulated the ex-pression and fluorescence intensity of M2 macrophage markers CD206 and ArgI proteins(P<0.05).With the extension of cell culture time,the proliferation activity of T24 cells in each group showed an increasing trend(P<0.05).Compared with the Control-NC group,SPHK1 overexpression increased the proliferation,migra-tion,and invasion abilities of T24 cells,while SPHK1 knockdown decreased the proliferation,migration,and invasion abilities of T24 cells(P<0.05).SPHK1 overexpression increased the expression of Vimentin and N-Cadherin proteins and decreased the expression of E-Cadherin protein in T24 cells(P<0.05).SPHK1 knockdown decreased the expression of Vimentin and N-Cadherin proteins and increased the expression of E-Cadherin protein in T24 cells(P<0.05).Conclusion:Overexpression of SPHK1 can promote the polarization of M2 macrophages,promote the proliferation,migration,invasion,and EMT of bladder cancer cells,and ul-timately increase the distant metastasis ability of bladder cancer.This finding is worthy of further clinical re-search.
万方数据
维普
