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目的:观察Y-盒结合蛋白1(Y-box binding protein-1,YB-1)对乳腺癌MCF-7细胞CD44表达及细胞迁移、成球能力的影响。方法:应用基因重组技术将YB-1基因及其siRNA构建于腺病毒载体pADeasy-1,重组腺病毒及腺病毒空载体转染乳腺癌细胞株MCF-7;通过蛋白质印迹法,RT-PCR和免疫荧光的方法检测YB-1和CD44的表达;MTT法检测转染后细胞生长速度,并绘制生长曲线;并通过Transwell小室法检测细胞侵袭和迁移能力;细胞使用无血清培养基培养,并加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)后,观察YB-1对细胞成球能力的影响。结果:蛋白质印迹法和RT-PCR结果显示,转染YB-1重组腺病毒载体的MCF-7细胞中,YB-1和CD44的表达明显高于转染腺病毒空载体组和未转染组;相反,转染YB-1 siRNA重组腺病毒载体的MCF-7细胞中,YB-1和CD44表达明显低于对照腺病毒空载体组和未转染组;免疫荧光也证实CD44蛋白水平与YB-1表达正相关。细胞生长曲线表明,转染YB-1重组腺病毒载体组的细胞生长速度最快,而且其侵袭、迁移和成球能力最强。结论:YB-1可能通过调控CD44的表达影响人乳腺癌细胞MCF-7的侵袭及成球能力。
OBJECTIVE: To observe the effect of Y-box binding protein-1 (Y-box binding protein-1) on CD44 expression, cell migration and spheroidization in breast cancer MCF-7 cells. Methods: YB-1 gene and its siRNA were constructed by adenovirus vector pADeasy-1 and recombinant adenovirus vector and transfected into breast cancer cell line MCF-7 by using gene recombination technique. Immunofluorescence method was used to detect the expression of YB-1 and CD44. The cell growth rate was detected by MTT assay and the growth curve was drawn. The cell invasion and migration ability was detected by Transwell chamber method. The cells were cultured in serum-free medium and added Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), to observe the YB-1 on the ability of the cells into the ball. Results: The results of Western blot and RT-PCR showed that the expression of YB-1 and CD44 in MCF-7 cells transfected with YB-1 recombinant adenovirus was significantly higher than that in transfected Adenovirus empty vector and untransfected group ; On the contrary, YB-1 and CD44 expression in MCF-7 cells transfected YB-1 siRNA recombinant adenovirus vector was significantly lower than the control adenovirus empty vector group and non-transfected group; immunofluorescence also confirmed CD44 protein levels and YB -1 expression was positively correlated. The cell growth curve showed that the cells transfected with YB-1 recombinant adenovirus had the fastest cell growth rate and the strongest ability of invasion, migration and spheroidization. Conclusion: YB-1 may affect the invasion and the ability of forming human breast cancer cell line MCF-7 by regulating the expression of CD44.