Effects Of Bone Marrow Stromal Cells HS-5 On Apoptosis Of U937 Cells By SiRNA Interference With Gli Gene

Objective:Research shows that HS-5 can protect the bone marrow stromal cells in leukemia cells,but the mechanism is not clear,investigated the effect of HS-5 on the apoptosis of acute myeloid leukemia(U937)cells co-cultured with HS-5 cells by using si RNA interference in the Hedgehog gene of U937 cells.Methods: si RNA-Gli-1 was transfected into U937 cell line by lipo2000 technique.After transfection,the cells were co-cultured in the pre-cultured HS-5 cells to simulate the effect of bone marrow microenvironment on leukemia cell apoptosis.Morphologicalchanges of si RNA-Gli-1 transfected cells were observed by Giemsa staining.Western blot and q RT-PCR were used to detect the expression of Gli-1 protein and Gli-1 m RNA after Gli-1-si RNA transfection.The proliferation of U937 cells was detected by CCK8 kit.The apoptosis rate of U937 cells was detected by Annexin V-PE and 7-AAD double staining flow cytometry.The apoptosis rate of U937 cells was detected by flow cytometry.PI was detected by flow cytometry.The expression of Bcl-2,Bcl-xl and Caspase3 in U937 cells were detected by q RT-PCR.Results: After transfection for 48 hours,the morphology of U937 si RNA-Gli-1 cells co-cultured with HS-5 cells under light microscope was slightly smaller and the light transmittance decreased slightly,showing little particles;Giemsa staining showed that with HS-5 cells The expression of Gli-1 protein and Gli-1 m RNA in U937 si RNA-Gli-1 cells were observed.The expression of Gli-1 protein and Gli-1 m RNA in U937 si RNA-Gli-1 cells were significantly higher than those in HS-5 cells.Cell proliferation is slow.U937 si RNA-Gli-1 cells were co-cultured with HS-5 cells for 48 hours.The U937 si RNA-Gli-1 cells co-cultured with HS-5 cells were blocked in G0 / G1 phase and co-cultured with HS-5 cells The expression of Bcl-2 and Bcl-xl m RNA in U937 si RNA-Gli-1 cells was significantly down-regulated.The m RNA expression of Caspase3 in U937 si RNA-Gli-1 cells co-cultured with HS-5 cells was significantly up-regulated.Conclusion: 1.Transfection of si RNA-Gli-1 into U937 cells with lipo2000 technique could down-regulate Gli-1 m RNA and protein expression in U937 cells.2.The expression of Bcl-2 and Bcl-x1 in U937 si RNA-Gli-1 cells co-cultured with U937 si RNA-Gli-1 cells was higher than that of U937 si RNA-NC cells co-cultured with HS-5 cells,and the expression of Caspase3 high.3.Interfering with Gli-1 gene can reverse the protective effect of bone marrow stromal cells HS-5 on acute myeloid leukemia cells U937.Part of the reason is through the regulation of Bcl-2,Bcl-xl twoanti-apoptotic gene and Caspase3 pro-apoptotic gene to achieve.