EPCR对人乳腺癌MDA-MB-231细胞增殖、迁移的影响及机制

Effects of EPCR on the proliferation and migration of human breast cancer cells MDA-MB-231

目的探讨内皮细胞蛋白C受体(EPCR)对乳腺癌MDA-MB-231细胞增殖、迁移的影响及机制。方法将MDA-MB-231细胞分为EPCR转染组、无关序列转染组和对照组,分别加入EPCR-siRNA、siRNA-NC、空脂质体转染,采用Cell-ELISA法检测EPCR对蛋白酶活化受体1(PAR-1)活性的影响、CCK-8法检测细胞增殖能力、Transwell法检测细胞迁移能力。添加抗体阻断PAR-1作用后,采用CCK-8和Transwell法分别检测细胞增殖及迁移能力。Western blotting法检测Erk1/2和AKT通路蛋白表达。结果与对照组及无关序列转染组相比,EPCR转染组未裂解活化的PAR-1抗体结合率升高(P<0.05),且EPCR转染组细胞增殖及迁移能力均降低(P均<0.05)。与对照组相比,PAR-1抗体处理后细胞增殖迁移能力均降低(P均<0.05)。EPCR转染组和抗体处理组细胞p-AKT(S473)和p-AKT(T308)蛋白表达均低于对照组(P均<0.05),而Erk1/2蛋白磷酸化与对照组比较差异无统计学意义(P>0.05)。结论 EPCR可依赖于PAR-1的裂解活化,然后通过活化AKT通路而影响人乳腺癌细胞MDAMB-231的增殖和迁移。

Objective To explore the effects and mechanism of endothelial protein C receptor(EPCR)on the proliferation and migration of human breast cancer cells MDA-MB-231.Methods MDA-MB-231 cells were divided into the EPCR transfection group,unrelated sequence transfection group,and control group which were transfected with EPCR-siRNA,siRNA-NC,and empty plasmid,respectively.Cell-ELISA was used to detect the activation of PAR-1 in the membranes of MDA-MB-231.CCK-8 assay was performed to observe the proliferation of cells in each group.Transwell chamber was employed to observe the cell's migration.After treatment of anti-PAR-1 antibody,the proliferation and migration abilities of MDA-MB-231 cells were detected by CCK-8 and Transwell assay.Western blotting was used to determine the protein expression levels of Erk1/2 and AKT.Results Compared with the control group and unrelated sequence transfection group,the binding rate of un-cleaved activated PAR-1 antibody increased,and the proliferation and migration abilities significantly decreased in the transfection group(all P<0.05).Compared with the control group,after being treated with anti-PAR-1 antibody,the proliferation and migration of MDA-MB-231 cells significantly decreased(both P<0.05).After knockdown of EPCR or the PAR-1 antibody treatment,the expression of p-Akt(S473)and p-Akt(T308)significantly decreased in the MDA-MB-231 cells(both P<0.05),but the expression of p-Erk1/2 did not change significantly(P>0.05).Conclusion EPCR contributes to the proliferation and migration of MDA-MB-231 cells by activating AKT,and this effect of EPCR may be dependent on PAR-1.