The Effect Of Human Tissue Factor Pathway Inhibitor-2 On The Expression Of VEGF In Multiple Myeloma

Tissue factor pathway inhibitor-2(TFPI-2) is a newly discovered serine protease inhhibitor with Kunitz-type structure,which is synthesized and secreted into extracellular matrix by vascular endothelial cells that mainly constituted vascular, smooth muscle cells and fibroblasts.TFPI-2 can strongly inhibit plamin,trypsin, chymotrypsin and other proteolytic activity in vitro and inhibit the original matrix metalloproteinase proMMP-1 and proMMP-3 activation which are catalyzed by plaminogen,but our previous study confirmed that it has no direct inhibitory effect on matrix metalloproteinase(Matrix Metalloproteinases,MMPs) such as MMP-1.Our study also found that some mammalian bladder epithelium can secrete TFPI-2,but its significance is still unknown.It has confirmed that extracellular matrix(ECM) degradation by tumor cell invasion and metastasis are a key step in the process,and plasminogen,trypsin and chymotrypsin and other serine protease secreted by tumor cell play an important role in ECM degradation.It has shown that fibroblastoma cells transfected by TFPI-2 gene in SCID mice significantly grow slower than non-transfected tumor cells,and led to the decline in the rate of metastasis in lung.It is meaningful that vascular endothelial growth factor (VEGF) mRNA expression levels in the tissue of transfected fibroblastoma cells with TFPI-2 gene were 3-6 times lower than in the non-transfected fibroblastoma cells. VEGF is one of the angiogenesis positive regulation factors,which plays a specific role in the vascular endothelial cell mitogen and stimulates vascular endothelial cell proliferation and migration,and promotes the growth of new blood vessel.The above results indicate that TFPI-2 inhibited in vivo tumor growth and distant metastasis and this is relevant to its inhibition of the expression of VEGF which reduced tumor angiogenesis.Since then researchers have found that the angiogenesis of transfected TFPI-2 glioblastoma cells was inhibited in vivo and the capillary-like structure was reduced in vitro.These findings indicated that TFPI-2 may be involved in the regulation of tumor angiogenesis.Recently,the relationship of TFPI-2 and VEGF has been further studied.Real-time quantitative PCR and Western blot analysis revealed that VEGF increased the expression of TFPI-2 mRNA and TFPI-2 protein in time and dose-dependent manner.In contrast,TFPI-2 protein inhibited VEGF-stimulated endothelial cell proliferation and VEGF-induced endothelial cell migration in a dose-dependent manner.The results revealed TFPI-2 may be involved in the regulation of VEGF in a negative feedback mechanism.TFPI-2 inhibited the expression of VEGF in some entity tumors and VEGF-dependent vascular endothelial proliferation and migration.TPFI-2 has the potential effect on anti-angiogensis.Multiple myeloma(MM) is one of the common malignant tumors,which is a serious threat to the health of middle-aged and elderly people.In recent years it has attracted popular attention because of increased incidence and limited treatments.The pathogenesis of MM is complex,and angiogensis is important in the occurrence and development of MM..VEGF and basic fibroblast growth factor(bFGF) producing from myeloma,bone marrow stromal cells(BMSCs),endothelial cells(ECs) together participate in angiogenesis in multiple myeloma.Studies show that the serum VEGF levels of patients with MM are inceaesed and relevant to the stage and prognosis of the disease.Therefore,one of the effective measures is to decrease the VEGF levels and thus inhibits tumor angiogenesis.In our study,the TFPI-2 gene was transfected into U266 cells.We wanted to find the difference between the VEGF levels in culture medium of transfected TFPI-2 gene and mock-transfected cells and the difference between the VEGF levels in culture medium of transfected TFPI-2 gene and mock-transfected cells co-cultured with BMSCs.This will give a new idea for the therapy of MM.. Method1 TransfectionsU266 cells in logarithmic phase were inoculated in 24-well culture plates coated with fibronectin,5×10⁵ per well.After 24 hours culture,the plates were washed 2 times with serum-free RPMI-1640 medium.The pcDNA3.0/TFPI-2 recombinant expression vector and a simple expression vector pcDNA3.0 were transfected into U266 cells using transfection reagent,Lipofectmine 2000.The former was set to the experimental group,the latter was the control group.The transfected cells were screened by 200 mg/L,400 mg/L,600 mg/L,800mg/L G418-wide culture medium respectively.The optimal selected concentration was 600 mg/L.Positive clones were cultured with 400mg/l G418 in culture medium.A stable screening time is about six weeeks and then the cells were used in the following tests.2 Cells culture:â‘ Experimental and control group of U266 cells were inoculated into 48-well plates with 1×10⁶ cells/ml/well,and cultured in 1%FBS in RPMI-1640 medium for 72 hours.Then the supernatant was collected,1500 rpm to 5 minutes,and the supernatants stored in -20℃for VEGF concentration assay with ELISA and the cells were stored in -80℃for RT-PCR experiments.â‘¡After digesting,BMSCs were inoculated into 48-well plates with 1×10⁵cells/ml/well containing 10%FBS of DMEM culture medium.After 24 hours, the supernatant was discarded and the plate was washed 2 times with serum-free RPMI-1640.The experimental and control U266 cells were added in 2×10⁵ cells/ml/well in serum-free RPMI-1640 medium.The culture medium was collected after 48 hours,1500 rpm to 5 minutes,and the supernatant was stored in -20℃for VEGF concentration assay with ELISA.3 RQ-PCR for TFPI-2 mRNATo set up Taqman Probe real-time quantitative RT-PCR technology to detect and quantify the TFPI-2 mRNA levels in transfected TFPI-2 gene and the control group cells.Independent-Samples T test was used to testify statistic meaning of VEGF levels between the two groups,testing the standardα=0.05.4 ELISA for VEGFAccording to human VEGF ELISA detection kit instuctions to carry out operations,we determined the VEGF levels in culture medium of transfected TFPI-2 gene and the control group cells and transfected TFPI-2 gene and the control group cells co-cultured with BMSCs.The TFPI-2 gene transferred factor and cultured with BMSCs factor have no interaction,Then independent-Samples T test was used to testify statistic meaning of VEGF levals between the two group,testing the standardα=0.05.5 RQ-PCR for VEGF mRNATo set up SYBR Greenâ… RQ-PCR technology to detect and quantify the VEGF mRNA levels in transfected TFPI-2 gene and the control group cells.Using formula: Folds=2^{(-△△Ct)},we analysis the relative changes in VEGF mRNA levels. Independent-Samples T test was used to testify statistic meaning of VEGF levels between the two groups,testing the standardα=0.05.ResultsThe TFPI-2 mRNA levels in transfected TFPI-2 gene and mock-transfected cells were significantly different,and the former was significantly higher than the latter (20.4440±15.72140 vs 0.7549±0.49670,P＜0.05).This indicated that TFPI-2 expression vector was transfected into U266 cells successfully.The VEGF levels in culture medium of transfected TFPI-2 gene and mock-transfected cells were not significantly different(86.75±7.76 ng/ml vs 91.53±11.58ng/ml,P＞0.05).The VEGF levels in culture medium of transfected TFPI-2 gene and mock-transfected cells co-cultured with BMSCs were significantly different,and the former was significantly lower than the latter(7.66±0.25ng/ml vs 8.86±0.44ng/ml,P＜0.05). The VEGF mRNA levels in transfected TFPI-2 gene mock-transfected cells were not significantly different(2^{(-△△Ct)} were 0.6130±0.07159 vs 0.8270±0.08380,P＞0.05).ConclusionsWith co-culture of BMSCs and TFPI-2 transfected MM cells,the level of VEGF in medium were significantly decreased.By such a result,we could probably conclude that TFPI-2 gene would indirectly inhibit the VEGF production and secretion of BMSCs.