MK通过EPCR/PAR1通路促进乳腺癌MDA-MB-231细胞体外血管生成

Study of MK promoting breast cancer cell MDA-MB-231 angiogenesis through EPCR / PAR1 pathway in vitro

目的探讨中期因子(midkine,MK)在人乳腺癌MDA-MB-231细胞体外血管生成中的作用及其机制。方法采用shRNA干扰技术降低MDA-MB-231细胞MK表达,应用Western blot技术检测肿瘤细胞中内皮蛋白C受体(endothelial protein C receptor,EPCR)的表达;干扰MK和EPCR表达或通过抗体阻断活化蛋白酶激活受体1(protease-activated receptor 1,PAR1)作用后,制备肿瘤条件培养基作用于人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),通过CCK-8试剂盒检测HUVECs增殖、Transwell小室检测迁移以及Matrigel表面培养检测脉管形成能力。结果干扰MK表达后,EPCR表达随之降低。干扰MK和EPCR低表达后,HUVECs增殖、迁移及脉管形成能力均低于对照组(P<0.05),EPCR干扰组低于MK干扰组(P<0.05)。应用抗PAR1作用后,HUVECs增殖、迁移及脉管形成能力低于对照组和EPCR干扰组(P<0.05)。结论 MK可通过EPCR/PAR1通路促进乳腺癌MDA-MB-231细胞体外血管生成。

Purpose To observe the effects of midkine （ MK） on human breast cancer cell line MDA-MB-231 angiogenesis in vitro, and to explore its mechanism. Method shRNA interference was performed to silence the expression of MK in MDA-MB-231 cells, and Western blot was used to identify the expression of MK and EPCR. After MK and EPCR knockdown, or treated with anti protease-activated receptor 1 （PAR1） antibody, the culture medium of MDA-MB-231 cells were collected and the conditioned medium were pre-pared. Human umbilical vein endothelial cells （ HUVECs） were cultured with conditioned medium, and the endothelial cells proliferation was detected by CCK-8 assay, cell migration was detected by transwell method, vasculogenic activity was assessed by Matrigel-based tube formation assay. Results After knockdown of MK, the protein level of EPCR was decreased in MDA-MB-231 cells. Compared with control, knockdown of MK and EPCR decreased the proliferation, migration and angiogenesis ability of HUVECs significant-ly （P〈0. 05）, and the effect of EPCR knockdown group was stronger than MK knockdown group （P〈0. 05）. After treated with anti-PAR1 antibody, the proliferation, migration and angiogenesis ability of HUVECs were decreased compared with control and EPCR knockdown group （P〈0. 05）. Conclusion MK promotes human breast cancer cell line MDA-MB-231 angiogenesis through EPCR /PAR1 signaling pathway in vitro.