The Influence Of TFPI-2 On The Growth And The Invasion Of K562 Cells

[Objective]Tissue factor pathway inhibitor-2(TFPI-2), previously designated as placental protein 5. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth, metastasis. In our study, we aimed at the influence of TFPI-2 on the growth and the invasion of K562 cells which belongs to one of the leukemic cell lines.[Methods]1. Human TFPI-2 expression vector pcDNA3.0-TFPI-2 and pcDNA3.0 were separately transfected into K562 cells using the LipofectamineTM 2000 reagent according to the manufacture's instruction. With 600mg/L G418 sulfateing, three groups that divided into K562,K562-V,K562-T were analysed in the following tests.2. Three groups cells including the stably transfected cells were analysed for TFPI-2 mRNA by real-time quantitative RT-PCR and for TFPI-2 protein by Western blotting.3. Three groups of cells were plated in each 24-well plate at a density of 2×103 /well. The growth curve was depicted by the average quantity of three wells which were counted every 24 hours. And we counted the daily growth rate and the average growth rate.4. Colony-forming cell assays: Three groups of cells were plated in each 6-well plates at a density of 40, 80, and 120 cultured for ten days. After that we counted the cloning rate by the proportion of cluster.5. We used the Boyden chamber to examine the ability of three groups cells with invasion in vitro. Three groups cells passing through the membrane of Boyden chamber was counted as the basis assessing cells under high-power microscope.[Results]1. The result of real time PCR: Expression of TFPI-2 mRNA in three groups were separately 1.04±0.65,1.44±2.27,61.85±49.52 , using the independent sample t'Test of the spss13.0, we found the expression of TFPI-2 mRNA in K562-T was higher than the rest of the other two groups, P<0.05, it had statistical significance.2. The result of western blot: TFPI-2 protein were detected in the stable-transfected cells line K562-T, but not in the other two groups.3. Seeing from the growth curve,in the K562-T group,the cell growth curve was flatter than those of other two groups,the average growth rates of each day was lower than those of other two groups.4. The result of colony-forming cell assays: In the K562-T group, cell cloning efficiency was lower than those of other two groups, using the Mann-Whitney Test of spss13.0 , P<0.05, it had statistical significance.5. In vitro Matrigel invasion assays:the number of penetration were separately 33.67±4.51,28.67±3.51,16.33±4.51, the number of penetration of K562-T was obviously decreased compared with the other two groups, using the independent sample t Test of the spss13.0,P<0.05, it had statistical significance.[Conclusions]Expression of TFPI-2 can not only strongly inhibit the invasive ability of K562 cells, but also inhibit the growth of K562 cells. Therefore, we speculate that TFPI-2 may have anti-tumor effect. It can reduce the proliferation and invasion of the tumor cells.