Sp1 Promotes NK/TCL Invasion By Up-regulating VEGF/VEGFR2 And IGF-1/IGF-1R Signaling Activities

Objective This study is aimed to identify the expression pattern of Sp1 gene in natural killer/T-cell lymphoma(NK/TCL)cell lines and to explore the role of Sp1 in the regulation of lymphoma cell invasion.Methods RT-PCR,Real-time PCR,immunofluorescence and western blot were used to detect the expression of Sp1.NK/TCL cells were incubated with Sp1 inhibitor Mythramycin A(MIT),and the expression levels of Sp1,VEGF,VEGFR2,IGF-1 and IGF-1R were quantified by Real-time PCR.Western blot was applied to detect the protein levels of Sp1,VEGF,IGF-1R and MMP2.Transwell assay was conducted to investigate the effects of inhibition of Sp1 by MIT on cell invasion.Results Sp1 was identified over-expression in NK/TCL cell lines as compared with that of NK cells from healthy donors.Expression levels of Sp1 m RNA in SNK-1,SNK-6 and SNT-8 were 8.4±0.28,12.6±0.12 and 7.6±0.24 folds increase as compared with that of control group,respectively.Levels of Sp1 protein were 5.6±0.44,10.0±0.93 and 7.6±0.23 folds increase times than control groups,respectively.Inhibition of Sp1 by MIT(100n M)significantly reduced cell invasion rate by 27.1±1.5%,31.1±1.8% and 31.8±1.7% as compared with controls.Inhibition of Sp1 by MIT(100n M)remarkably reduced expression levels of VEGF m RNA to 25.0±1.0%,31.3±2.7% and 38.5±3.1% as compared with controls.Meanwhile,levels of VEGF protein were reduced to 58.8±2.1%,55.8±1.8% and 46.2±2.9% as compared with controls.Inhibition of Sp1 by MIT(100n M)showed no significant effect on the expression of VEGFR2.MIT at 100 n M also reduced expression levels of IGF-1R m RNA in SNK-1 and SNK-6 cells,while not in SNT-8,which has been found no expression of IGF-1R.IGF-1R m RNA levels in SNK-1 and SNK-6 cells were decreased to 16.1±3.7% and 34.2±4.2% of control groups,and levels of IGF-1R protein were reduced to 48.5±7.1% and 50.4±9.1% of control groups.Inhibition of Sp1 by MIT showed no significant effect on the expression of the ligand of IGF-1.MMP2,a key protein in the regulation of cell invasion,was also significantly reduced expression in the presence of MIT in these three cell lines.The protein levels were found to be 70.7±4.7%,49.7±5.7% and 47.2±4.9% of controls.Conclusion Sp1 is over-expressed in all the three NK/TCL cells.Sp1 may promotes lymphoma cells invasion by up-regulating VEGF and IGF-1R expression,which enhance VEGF/VEGFR2 and IGF-1/IGF-1R signaling.MMP2 is also involved in process of Sp1-induced cell invasion in NK/T lymphoma cells...