SOX2 And Chemotaxis-related Genes In Human Myeloma Cell Line H929 Modulated By Interleukin-1β Treated Human Bone Marrow Derived Mesenchymal Stem Cells

OBJECTIVE: To investigate the effects of interleukin-1β(IL-1β) treatment on the osteogenetic potentials of human bone marrow mesenchymal stem cells(BMMSCs), and also to inverstigate the IL-1β primed BMMSCs’ ability to modulate both stem cell-associated proteins expression and chemotaxis-related receptors transcription of myeloma cells when two types of cells were co-cultured.METHODS: 1、There are three cohorts in this experiment: untreated normal bone marrow BMMSCS served as control(cohort A); BMMSCs only treated by IL-1β once(cohort B); BMMSCs were treated once a day for seven continuous days(cohort C). Then treated or untreated BMMSCS was employed as coating feeder for the directed coculture with H929 cells for three days.At the end of co-culture,H929 myeloma cells were harvested for subsequent assays. 2 、 The levels of bone morphgenetic protein-2(BMP-2) in BMMSCs were measured by Immunohistochemistry. NF-k B inhibitor( Pyrrolidine dithiocarbamate, PDTC) was sometime added before IL-1β treatment. 3、After co-cultured with BMMSCs, the total protein of H929 were extracted. The expression of NANOG, SOX2 and OCT-4 was measured by western blot. 4、Total RNA of the H929 cells were extracted after co-culture.The m RNA levels of CCR1, CCR2, CCR6, CXCR4, MCP-1, MIP-1α and DKK1 were measured by real-time PCR.RESULTS: 1、The in vitro cultured BMMSCs were spindle-shaped and the H929 cells spherical shaped. The morphology and growth pattern of BMMSCs were not significantly different among the three cohorts. 2、Compared with cohorts A, BMP-2 protien levels were higher in both B and C cohorts, and the levels of BMP-2 protein in both IL-1β treated cohorts was remarkedly down-regulated in the presence of NF-k B pathway inhibitor PDTC.3、The SOX2 protein levels in H929 cells were higher in both B and C cohorts than that of A cohort, and there was statistical difference among cohorts(n=5; P<0.05). However, The protein levels of either NANOG or OCT-4 protein did not differ significantly among the three cohorts. 4、In the respective of chemokines and its receptors, the m RNA levels of CCR1, CCR2, MCP-1, MIP-1α of myeloma cells in C cohort were higher than those in either cohort A or B, and there was also a statistical difference among three cohorts( n=10; P<0.05). However, there were no statistical differences in the m RNA levels of CCR6, CXCR4 and DKK1 among three cohorts.CONCLUSION: Our in vitro results indicated that inflammatory cytokine IL-1β enhanced the levels of BMP-2 protein in BMMSCs at least patrially via NF-k B pathway. It also implied that the IL-1β in bone marrow microenvironment may also regulate myeloma cells indirectly via non-myeloma cells such as BMMSCS, besides, sustained elevated IL-1β level in bone marrow might has more profound effects on myeloma cells through BMMSCs. BMMSCs is an important cellular component in bone marrow microenvironment involved in myeloma progression.