沉默β-catenin表达对黑色素瘤A375细胞增殖与凋亡的影响研究

Effects of silencing β-catenin expression on proliferation and apoptosis of melanoma A375 cells

目的 探讨沉默Wnt/β-catenin信号通路中的关键蛋白β-catenin对人恶性黑色素瘤A375细胞增殖与凋亡的影响。方法 将干扰慢病毒液(β-catenin-RNAi)和空载体慢病毒液(β-catenin-negative)分别感染A375细胞,得到的稳定感染细胞株分别标记为A375-RNAi、A375-negative。采用Western blot法检测感染后的各组细胞中β-catenin蛋白表达水平,MTT法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率。结果 β-catenin在A375细胞中高表达,在A375-RNAi细胞中的表达水平下降约55.1%(P<0.05),即β-catenin被干扰慢病毒液有效沉默;A375-negative细胞β-catenin蛋白表达水平与A375细胞比较无统计学差异(P>0.05)。A375-RNAi细胞与A375-negative细胞、A375细胞相比增殖活性均减弱(均P<0.05);A375-negative细胞增殖活性与A375细胞比较无统计学差异(P>0.05)。A375-RNAi细胞与A375-negative细胞、A375细胞相比凋亡率明显增高(均P<0.05);A375-negative细胞凋亡率与A375细胞比较无统计学差异(P>0.05)。结论 慢病毒载体介导si RNA能有效沉默人黑色素细胞瘤A375细胞内β-catenin蛋白的表达。下调β-catenin表达能抑制A375细胞增殖,细胞凋亡率增高。

Objective To investigate the effect of silencing β-catenin expression on the proliferation and apoptosis of malignant melanoma A375 cells. Methods The β-catenin-RNAi lentivirus and β-catenin-negative lentivirus were constructed and transfected into A375 cells（A375-RNAi and A375-negative）. The expression of β-catenin protein was detected with Western blot; the cell proliferation was determined with MTT method; the cell apoptosis rate was examined with flow cytometry. Results Western blot showed that the protein expression of β-catenin in A375-RNAi cells was lower than that in A375-negative and A375 cells（P〈0.05）, while the expression levels in the latter two had no significant difference（P〉0.05）. MTT showed that compared with A375 cells the proliferative activity of A375-RNAi cells was reduced（P〈0.05）, and there was no significant difference between A375-negative cells and A375 cells（P〉0.05）. Flow cytometry showed that the apoptosis rate of A375-RNAi cells was significantly higher than that in A375 cells（P〈0.05）, and there was no significant difference between A375-negative cells and A375 cells（P〉0.05）. Conclusion Transfection with β-catenin-RNAi-LV can successfully down-regulate the expression of β-catenin, which leads to the inhibition of proliferation and enhancement of apoptosis in A375 cells.