蛋白酶活化受体1在B16F10瘤细胞体内外迁移中的作用

Study on the role of protease-activated receptor 1 in promoting B16F10 metastasis in vitro and in vivo

目的:研究蛋白酶活化受体 1(PAR1)在肿瘤细胞转移过程中的作用,评估凝血系统与肿瘤转移的关系。方法:首先通过逆转录聚合酶链反应(RT-PCR)和 Western-blot 方法检测了 siRNA 对 B16F10 黑色素瘤细胞中 PAR1的内源表达的影响,然后在 PAR1 受 siRNA 干扰的情况下检测 B16F10细胞在体内外迁移能力,其结果与未受siRNA 干扰的试验结果相对照,来确定 PAR1 在 B16F10 瘤细胞体内外迁移中的作用。结果:75nmol/L 的 siRNA 干扰48h 后可明显抑制 B16F10中 PAR1 的表达,其转录水平抑制率是60%,蛋白水平的抑制率为85%;体外迁移实验表明,PAR1 表达量减少后与对照组相比细胞迁移能力减弱(P<0.01);动物实验表明.干扰 B16F10细胞中的 PAR1后,肿瘤细胞经血管迁移能力减弱,在肺部形成的转移瘤数目减少(P<0.01)。结论:PAR1 具有促进肿瘤细胞迁移作用。开发 PAR1 封闭剂有望抑制肿瘤转移,可作为研制抗肿瘤转移药物的新靶点。

Objective To observe the effects of the protease-activated receptors （PAR1） on the metastasis of the murine melanoma cell line B16F10 in vivo and in vitro, and to study the close linkage between the malignancy and the coagulation. Methods Both reverse transcription polymerase chain reaction （RT-PCR） and Western-blot were used to test the effects of the small interfering RNA（siRNA） on inhibiting the PARI expression in the B16F10. The metastatic activity of the B16F10 with siRNA transfection was then tested both in vitro and in vivo. All the observations were compared with the counterparts without siRNA transfection to determine the roles of the PAR1 in the B16F10 metastasis. Results The PAR1 expression was efficiently reduced after 48 h while the cells were transfected with siRNA （75 nmol/L ）, and correspondingly the cell migration activity in vitro was significantly impaired （P〈0.01）. In vivo studies with C57/BL6 mice also showed that after the PAR1 expression was inhibited in the B16FlO, the migration activity in the blood vessels was greatly weakened. And in the pulmonary metastasis model, the number of the tumor loci significantly decreased. Conclusions The PAR1 can efficiently promote the cell migration activity. The results suggeste the potential to develop the therapeutic anti-metastasis drugs through blocking the PAR1.