摘要
目的研究PI3K p55γ调节亚基N末端氨基酸过表达对乳腺癌细胞MDA-MB-231黏附性的影响,探讨其作用的分子机制。方法通过脂质体介导用重组质粒p EGFPN24瞬时转染MDA-MB-231细胞系。荧光显微镜和免疫印迹方法鉴定转染细胞中绿色荧光蛋白(GFP)以及融合蛋白GFP-N24的表达,细胞黏附实验检测细胞在胶原和纤黏连蛋白上的黏附性。免疫印迹实验检测细胞内源性E-钙黏素(E-cadherin)和磷酸化Akt(p Akt)的表达。酶谱实验检测基质金属蛋白酶9(MMP9)和尿激酶型纤维酶原活化因子(u PA)的表达与分泌。结果荧光显微镜下可见转染细胞因外源基因的表达而发出绿色荧光,胞浆分泌颗粒明显增加。免疫印迹结果显示,融合蛋白GFP-N24的表达量略低于空载体中GFP的表达量。过表达N24p55γ的细胞在纤黏连蛋白和胶原上的黏附性明显下降。细胞内源性PI3K下游信号分子磷酸化Akt的表达量下降,但N24p55γ的过表达不影响转染细胞中细胞黏附分子E-cadherin的表达。此外,N24p55γ的过表达抑制肿瘤转移相关基因MMP9的活化与u PA的分泌。结论 PI3K p55γN末端氨基酸过表达可能通过影响PI3K/Akt信号通路以及肿瘤转移相关基因MMP9和u PA的表达抑制乳腺癌MDA-MB-231细胞的黏附性。
This study was performed to investigate the effects of overexpression of the N-terminal 24-aminoacid of p55γ regulatory subunit of phosphoinositide 3-kinase（PI3K） on cell adhesion of breast cancer cellMDA-MB-231. MDA-MB-231 cell lines expressing GFP-N24 fusion protein or green fluorescence protein（GFP）protein alone were generated by transient transfection with p EGFPN24 plasmid and control plasmid p EGFPC1,respectively. The expression of GFP and fusion protein GFP-N24 were observed by fluorescence microscope andWestern blotting. The adhesion of the transfected cells in collagen and fibronectin was detected by cell adhesionassay. The expression of E-cadherin and phospho-Akt were analyzed by Western blotting. The expressionand secretion of matrix metalloproteinase-9（MMP9） and urokinase plasminogen activator（u PA） were detected byzymography. Under fluorescence microscope, the green fluorescence was observed in the transfected cells, and thesecretory granules increased obviously. The results of Western blot showed that the expression level of the fusionprotein GFP-N24 was slightly lower than GFP expression in the empty vector. Compared with the control cells,the adhesion of MDA-MB-231 cells to fibronectin and collagen was decreased in N24p55γ over expressinggroup. Overexpression of N24p55γ decreased the expression of phosphor-Akt but not effected the expression ofE-cadherin in MDA-MB-231 cells. The activation and secretion of MMP9 and u PA were inhibited by GFP-N24 expression in MDAMB-231 cells. In conclusion, overexpression of N24p55γ inhibits cell adhesion of MDA-MB-231 by influencing PI3K/Akt signaling pathway and the expression of tumor metastasis associated gene u PA and MMP9.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2016年第4期315-319,共5页
Immunological Journal
基金
黑龙江省青年科学基金(QC2015120)
