Nup88通过钙调蛋白调控乳腺癌细胞侵袭能力

Nup88 affect the invasion ability of BT-20 cells by binding to calcium-binding protein

目的 探讨核孔蛋白88kDa(Nup88)基因影响乳腺癌细胞系BT-20细胞侵袭能力的分子机制。方法 敲低Nup88基因后,通过Transwell和划痕实验检测细胞的侵袭能力,通过TMT标记的定量蛋白质组技术,采用流式细胞术检测Nup88敲低细胞的钙离子水平,通过明胶酶谱实验检测Nup88敲低表达后,乳腺癌BT-20细胞分泌基质金属蛋白酶2(MMP2)和MMP9的情况。通过pull-down试验寻找并验证与Nup88存在相互作用的蛋白。结果 敲低Nup88基因表达后,细胞培养48h后,划痕愈合率低于对照组,穿膜细胞数量亦显著低于对照组,BT-20细胞的迁移能力减弱。蛋白质组学分析得到差异蛋白247个,其中Nup88基因敲低细胞株的Nup88蛋白表达水平降低约48%。GO分析表明,敲低Nup88的表达可能影响了钙结合相关蛋白的表达。细胞实验结果表明,细胞中钙离子含量显著降低,分泌MMP2和MMP9的活性显著低于阴性对照组细胞(P<0.05)。Pull-down结果证实Nup88与多个钙结合蛋白存在相互作用,可能通过二硫键与钙结合蛋白39(CAB39)相同作用,共同调控钙离子水平。结论 Nup88可能通过与CAB39结合,调节胞内钙离子含量,调节基质金属蛋白酶的活性,进而影响肿瘤细胞的侵袭能力。

Objective To investigate the molecular mechanism of(nucleoporin 88 kDa)Nup88 gene on invasion abil-ity of breast cancer cell line BT-20.Methods After the Nup88 gene is knocked down,the invasion capability of the cell is detected through Transwell and scratch experiments,the calcium ion level of the Nup88-knocked down cell is detected by flow cytometry through a TMT labeled quantitative proteome technology,and after the Nup88 knock-down expression is detected through a gelatin zymogram experiment.The secretion of matrix metalloproteinase 2(MMP2)and MMP9 by BT-20 cells.Proteins interacting with Nup88 were identified by pull-down assay.Results After knockdown of Nup88 gene expression,the wound healing rate was lower than that of the control group after 48 h of cell culture,the number of membrane-penetrating cells was also significantly lower than that of the control group,and the migration ability of BT-20 cells was weakened.Pro-teomic analysis showed that there were 247 differentially expressed proteins,and the expression level of Nup88 protein in Nup88 knockdown cell line was reduced by about 48%.GO analysis showed that knockdown of Nup88 expression may affect the expression of calcium-binding related proteins.The results of cell experiment showed that the content of calcium ion in the cells was significantly decreased,and the activities of MMP2 and MMP9 secreted by the cells were significantly lower than those of the negative control cells(P<0.05).Pull-down results confirmed that Nup88 interacted with several cal-cium-binding proteins,possibly through disulfide bonds with calcium-binding protein 39(CAB39)to regulate calcium lev-els.Conclusion Nup88 may bind to CAB39 to regulate the intracellular calcium content and the activity of matrix metallo-proteinases,thereby affecting the invasive ability of tumor cells.