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本课题组先前已证明NUAK1/ARK5可通过影响F-actin的聚合从而促进乳腺癌细胞的侵袭和转移.但是NUAK1是否还通过其它机制影响乳腺癌的侵袭和转移尚有待于探讨.本文证明NUAK1还可以影响乳腺癌细胞趋化、粘附能力从而在乳腺癌细胞侵袭转移中起重要作用.应用化学合成的小RNA干扰质粒转染到乳腺癌细胞系MDA-MB-231中,用免疫印迹技术检测NUAK1蛋白的表达情况.结果显示,在敲除NUAK1的细胞(siNUAK1/MDA231)中,NUAK1蛋白表达水平明显降低;趋化运动实验结果显示,siNUAK1/MDA231细胞的趋化运动能力比未处理组(Scr/MDA231)细胞明显降低;细胞粘附实验结果显示,EGF刺激5 min、15 min后,siNUAK1/MDA231细胞比Scr/MDA231细胞粘附细胞数量均明显减少;免疫印迹技术检测integrinβ1磷酸化验证NUAK1影响乳腺癌细胞粘附的机制.结果显示,siNUAK1/MDA231细胞内integrinβ1磷酸化比Scr/MDA231细胞不同程度降低.上述结果表明,NUAK1通过磷酸化integrinβ1促进乳腺癌细胞与纤维粘连蛋白的粘附,从而促进乳腺癌的侵袭和转移.
Our group has previously shown that NUAK1 / ARK5 can promote the invasion and metastasis of breast cancer cells by affecting the polymerization of F-actin.However, whether NUAK1 also affects the invasion and metastasis of breast cancer by other mechanisms remains to be explored.This paper demonstrates that NUAK1 Can affect breast cancer cell chemotaxis, adhesion and thus play an important role in the invasion and metastasis of breast cancer cells.Cytosomal RNA interference plasmids were transfected into breast cancer cell line MDA-MB-231 by using chemical synthesis and detected by Western blotting NUAK1 protein expression in NUAK1 knockout cells. The results showed that NUAK1 protein expression was significantly decreased in NUAK1 knockdown cells (siNUAK1 / MDA231). Chemotaxis test showed that the chemotaxis ability of siNUAK1 / MDA231 cells was significantly higher than that of untreated group Scr / MDA231 cells were significantly decreased. The results of cell adhesion assay showed that the number of adherent cells in siNUAK1 / MDA231 cells was significantly decreased compared with that of Scr / MDA231 cells after stimulated with EGF for 5 min and 15 min respectively. Immunoblotting detected integrinβ1 phosphorylation of NUAK1 Affect the mechanism of breast cancer cell adhesion.The results showed that the phosphorylation of integrinβ1 in siNUAK1 / MDA231 cells decreased to a certain extent than that of Scr / MDA231 cells.The above results Show that NUAK1 promotes the invasion and metastasis of breast cancer through the phosphorylation of integrinβ1 to promote the adhesion of breast cancer cells and fibronectin.