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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。
AIM: To investigate the effect of mi R-203 on the invasion and migration of breast cancer cell line MDA-MB-231 by NEDD9. METHODS: The mi R-203 liposome was transfected into MDA-MB-231 cells. The cell invasion and migration were observed by cell scratch assay and Transwell assay. The effect of mi R-203 on NEDD9 expression was detected by Western blotting. And the reporter site of mi R-203 was determined by sensor reporter. Finally, the expression of related proteins was studied by “cell rescue” experiment and the changes of lamellipodia and focal adhesions were observed by immunofluorescence confocal laser scanning microscopy To investigate the regulatory mechanism of mi R-203 on the invasion and migration of MDA-MB-231 cells. Results: The cell scratch assay and Transwell assay showed that mi R-203 inhibited the invasion and migration of MDA-MB-231 cells. The bioinformatics website predicted that the target gene of mi R-203 was NEDD9. Western blotting and reporter The results showed that mi R-203 down-regulated NEDD9 by binding to NEDD9 3’-UTR; cell scratch assay, Western blotting and immunofluorescence after “rescue” experiments co-transfected with si NEDD9 and mi R- Confocal laser scanning results showed that mi R-203 inhibited the expression of NEDD9, resulting in the decrease of Rac1-GTP in activation state, which resulted in the change of cell motility and the invasion and migration ability, while the interference of si NEDD9 could “rescue” mi R- Inhibition of Migration Capacity of MB-231 Cells. CONCLUSION: mi R-203 can down-regulate the level of activated Rac1 through the degradation of NEDD9, leading to the decrease or disappearance of lamellipodia and focal adhesions in breast cancer cells MDA-MB-231, thereby inhibiting cell invasion and migration.