Notch3信号通路对三阴性乳腺癌细胞株BT20活性的影响及分子机制

Effect of Notch3 signaling pathway on BT20 activity of triple-negative breast cancer cell line and its molecular mechanism

目的探讨Notch3信号通路对三阴性乳腺癌细胞株BT20活性的影响及分子机制。方法采用小干扰RNA(siRNA)转染技术,下调BT20细胞中Notch3 mRNA表达,MTT法检测转染后5 d的细胞增殖情况,并进行Transwell细胞迁移和侵袭实验;RT-PCR检测BT20细胞中CyclinD1、BCL-2、BCL-xlmRNA表达水平;Western blot检测BT20细胞核内NF-κB蛋白表达水平。结果培养第2天、第3天、第4天、第5天时,Notch3 siRNA组细胞增殖水平明显低于空白对照组与阴性对照组,差异均有统计学意义(t分别=3.15、4.03、2.94、3.26、4.01、3.50、2.97、3.11,P均<0.05);随着培养时间的延伸,三组细胞增殖水平均逐渐增加,差异均有统计学意义(F分别=5.94、7.65、9.76,P均<0.05)。Notch3 siRNA组迁移细胞数、侵袭细胞数均明显低于空白对照组和阴性对照组,差异均有统计学意义(t分别=5.32、6.02、4.97、5.07,P均<0.05);空白对照组与阴性对照组迁移细胞数、侵袭细胞数比较,差异均无统计学意义(t分别=0.39、0.57,P均>0.05)。Notch3 siRNA组细胞核内NF-κB蛋白相对表达量明显低于空白对照组和阴性对照组,差异均有统计学意义(t分别=19.69、18.07,P均<0.05)。Notch3 siRNA组下游基因CyclinD1、BCL-2、BCL-xlmRNA的相对表达量均明显低于空白对照组和阴性对照组,差异均有统计学意义(t分别=4.15、3.02、3.12、2.98、3.56、3.16,P均<0.05)。结论Notch3 siRNA下调Notch3表达能够有效抑制BT20细胞增殖、迁移和侵袭,其机制可能与Notch3表达下调降低了NF-κB蛋白及CyclinD1、BCL-2、BCL-xl基因表达有关。

Objective To investigate the effect of Notch3 signaling pathway on the activity of triple negative breast cancer cell line BT20 and its molecular mechanism.Methods Small interfering RNA(siRNA)transfection technique was used to down regulate the expression of Notch3 mRNA in BT20 cells.MTT assay was used to detect cell proliferation 5 days after transfection,and Transwell cell migration and invasion experiments were performed.The expression levels of CyclinD1,BCL-2 and BCL-xl mRNA in BT20 cells were detected by RT-PCR.Detection of NFκB protein expression level in BT20 nuclei by Western blot.Results On the second,third,fourth and fifth days after culture,the cell prolifera-tion level of Notch3 siRNA group was significantly lower than that of blank control group and negative control group(t=3.15,4.03,2.94,3.26,4.01,3.50,2.97,3.11,P<0.05).With the extension of culture time,the cell proliferation level of the three groups increased gradually,and the difference was statistically significant(F=5.94,7.65,9.76,P<0.05).The number of migrating cells and invasive cells in the Notch3 siRNA group were significantly lower than those in the blank control group and the negative control group(t=5.32,6.02,4.97,5.07,P<0.05).There was no significant difference in the number of migrating cells and invasive cells be-tween the blank control group and the negative control group(t=0.39,0.57,P>0.05).The relative expression of NF-κB protein in the nucleus of Notch3 siRNA group was significantly lower than that of blank control group and negative control group(t=19.69,18.07,P<0.05).The relative expressions of CyclinD1,BCL-2,BCL-xl mRNA in the Notch3 siRNA group were significantly lower than that in the blank control group and the negative control group(t=4.15,3.02,3.12,2.98,3.56,3.16,P<0.05).Conclusion Downregulation of Notch3 expression by Notch3 siRNA can effectively inhibit the proliferation,migration and invasion of BT20 cells.The mechanism may be related to the downregulation of Notch3 ex-pression which reduces NFκB protein and CyclinD1,BCL-2,BCL-xl gene expression.