miR-203调控NEDD9抑制乳腺癌细胞MDA-MB-231侵袭及迁移作用机制研究

Study on mechanism of mi R-203 inhibiting migration and invasion of breast cancer cell MDA-MB-231 by regulating NEDD9 protein

目的 :研究mi R-203抑制乳腺癌MDA-MB-231细胞NEDD9蛋白的作用对细胞侵袭和迁移的影响。方法:mi R-203脂质体转染MDA-MB-231细胞,通过细胞划痕实验和Transwell实验观察细胞侵袭和迁移能力的变化;通过蛋白印迹检测mi R-203对NEDD9表达的调控作用,并用sensor reporter确定mi R-203的靶标位点;最后,通过细胞"拯救"实验研究相关蛋白表达量的变化,用免疫荧光-激光共聚焦显微镜观察细胞片状伪足和黏着斑的改变,探究mi R-203对MDA-MB-231细胞侵袭和迁移能力的调节机制。结果:细胞划痕实验和Transwell实验显示,mi R-203抑制了MDA-MB-231细胞的侵袭和迁移能力;通过生物信息学网站预测mi R-203的靶基因是NEDD9;蛋白印迹和sensor reporter检测结果显示,mi R-203通过与NEDD9 3′-UTR结合,下调NEDD9;共转染si NEDD9和mi R-203的"拯救"实验后的细胞划痕实验、蛋白印迹和免疫荧光-激光共聚焦结果显示,mi R-203抑制NEDD9表达导致激活态Rac1-GTP减少,致使细胞运动状态改变,侵袭迁移能力减弱,而si NEDD9干涉作用能"拯救"mi R-203对MDA-MB-231细胞迁移能力的抑制。结论 :mi R-203通过降解NEDD9,下调激活的Rac1水平,导致乳腺癌细胞MDA-MB-231片状伪足和黏着斑的减少或消失,从而抑制细胞的侵袭及迁移。

Object： To investigate the inhibiting effect of mi R-203 on invasion and migration of breast cancer cell MDA-MB-231 via inhibiting NEDD9 protein. Methods： mi R-203-liposime was transfected into MDA-MB-231 cells. Capacity of cell invasion and migration was assessed by cell scratch assay and Transwell migration assay. Western blotting was used to detect the expression of NEDD9, and sensor reporter assay was used to identify the targeting site of mi R-203.In cell ＂rescue＂ test, changes of related protein expressions were determined, and alterations of lamellipodium and focal adhesion were observed with immunofluorescence-laser scanning confocal microscopy. Results： Cell scratch assay and Transwell migration assay showed that the capacity of invasion and migration was inhibited by mi R-203. By bioinformatics website, NEDD9 was predicted as the target gene of mi R-203. Western blotting and sensor reporter assay showed mi R-203down-regulating NEDD9 via binding with NEDD9 3′-UTR. In rescue experiment, MDA-MB-231 cells were co-transfected with mi R-203 and si NEDD9. The reduction of cell invasion and migration capacity was due to the reduction of activated Rac1-GTP because of the reduction of NEDD9 due to mi R-203 inhibition. si NEDD9 could rescue the reduction of invasion and migration capacity. Conclusions： mi R-203 down-regulates the activated Rac1 through decreasing NEDD9, resulting in decrease of lamellipodium and focal adhesion, thereby inhibiting the invasion and migration of MDA-MB-231 cells.