PI3Kγ regulates tumor-associated macrophage phenotype and influences EMT pro-gression and stem characteristics of cholangiocarcinoma cells
Objective To explore the effect of phosphatidylinositide 3-kinase γ(PI3Kγ)on the EMT process and dry characteristics of cholangiocarcinoma cells by regulating tumor-associated macrophage(TAM)phenotype.Methods Phorbol 12-myristate 13-acetate(PMA)and IL-4 were used to induce the establishment of M2-type TAM,and at the same time,PI3Kγ inhibitor AS605240 was used to intervene.The cell morphology changes were observed,and immunocytofluorescence staining was used to detect the expression of M2-specific phenotype marker CD163 on the cell surface.A co-culture system of M2-type TAM and human cholangiocarcinoma cell line QBC-939 was established,and the cells were divided into control group,M2 group and PI3Kγ inhibitor group.After corresponding treatment,QBC-939 cells were collected.Transwell assay was used to detect cell migration and invasion ability,and immunocyt-ofluorescence staining was used to observe the expressions of E-cadherin and Vimentin in cells.Western blotting was used to detect the protein expression of E-cadherin,N-cadherin,Vimentin,MMP2 and MMP9 in cells,and tumor cell spheroidization assay was used to detect cell stemness.Western blotting was used to detect the expression of cancer stem cell-related proteins CD133,OCT4 and SOX2 in the cells.Results After induction by PMA and IL-4,the cells showed a typical M2 shape,and the CD163 fluorescence intensity increased significantly.However,after the intervention of PI3Kγ inhibitor,the number of M2 cells was significantly reduced,and the fluorescence intensity of CD163 was weak-ened.Compared with the M2 group,the number of cells migrated and invaded in the PI3Kγ inhibitor group decreased(P<0.05).The fluorescence density of E-cadherin increased,the fluorescence density of Vimentin decreased(P<0.05),and the expression level of E-cadherin protein was up-regulated.However,the protein expressions of N-cadherin,Vimentin,MMP2 and MMP9 were all down-regulated(P<0.05),at the same time,the number of tumor cells sphe-roids decreased(P<0.05),and the protein expressions of CD133,OCT4 and SOX2 were down-regulated(P<0.05).Conclusion Inhibition of PI3Kγ can inhibit the phenotypic transformation of M2 TAM,thereby inhibiting the EMT process and tumor stemness characteristics of cholangiocarcinoma cells,and exerting an anticancer effect.
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维普
