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Effects of TF-siRNA on the level of TF expression, proliferation, apoptosis and tube formation in the in vitro model of CNV |
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| Effects of TF-siRNA on the level of TF expression, proliferation, apoptosis and tube formation in the in vitro model of CNV |
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作者：彭文艳 文章来源：南通大学附属医院眼科 点击数：310 更新时间：2012/9/13 
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Background Choroidal neovascularization (CNV) is a major pathological feature at the advanced stage of age-related macular degeneration (AMD), pathological myopia and many other eye diseases. Recently many studies demonstrate that TF plays a key role in the processes underlying the pathogenesis of CNV. This study aimed to establish whether TF-siRNA (small interfering RNA targeting tissue factor) can inhibit/knockdown TF gene expression in the HUVECs line after LPS stimulation, which is widely used as an in vitro model of CNV, specificity and effectively, and therefore whether it might be used to influence cell proliferation, cell apoptosis, and tube formation.
Methods Lipopolysaccharide (lipopolysaccharide, LPS) stimulates HUVEC cells to simulate CNV (choroidal neovascularization) model in vitro. Initially we choose an optimal concentration of LPS to stimulate HUVEC cells, and screen a most effective TF-siRNA. 3 TF-siRNAs（they are TF-siRNA 1. TF-siRNA 2 and TF-siRNA 3.）, 1 VEGF-siRNA and 1 nonspecific-siRNA (NC) have designed (the siRNAs are all from biological technology Co., LTD). HUVECs stimulated by LPS were transfected with siRNAs above respectively for 24h, mRNA and protein expression of TF in HUVECs were evaluated by real-time RT-PCR and Western blot analysis. The effects on cell viability, apoptosis and tube formation treated with siRNAs, were examined in vitro by MTT assay, wound-healing assays, FACS and Matrigel-induced capillary tube formation.
Results 10 μ g/ml LPS has no influence on the cell vitality tested by MTT, Treatment with LPS (10 μg/ml) potently induced the expression of TF values at 24h in HUVECs, 5 times higher than untreated group. In the in vitro model of CNV, the level of TF mRNA was inhibited by about 40.5%. 78.9% and 16.0% after transfected with TF-siRNA 1. TF-siRNA 2 and TF-siRNA 3. respectively. siRNA treatment consistently down-regulated TF transcript and protein expression, the effect of TF-siRNA 2 is significant compared with the blank control (P <0.001). And therefor we used TF-siRNA 2, the most particular effective in the three sets of TF siRNAs, for the next experiment. MTT and Wound-healing assays show that the TF-siRNA 2 can inhibit the vitality of HUVEC cells evidently compared with the untransfected group. FACS showed that TF-siRNA 2 can also induce apoptosis about 4.07% and 9.16% after transfected at the final concentration of 50nm/l and 100nm/l. respectively. compared with the blank control about 1.03%. The effect of the Matrigel-induced capillary tube formation inhibition is also significant, decreased by 47.4% and 59.4% following treatment with the TF siRNA 2 and VEGF siRNA compared with the blank control (P <0.001), respectively.
Conclusion This study demonstrated that the TF-siRNA can inhibit or knockdown the expression of TF in HUVECs and therefore to suppress HUVEC cell proliferation, wound-healing ability, induce apoptosis, and can also inhibit angiogenesis in vitro. And above all knockdown of TF expression by TF-siRNA may provide a novel therapeutic target for the treatment of choroidal neovascularization-related diseases. |
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